Faculty Bibliography

Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high-resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate oncogenes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. These studies also suggested that CCND1 at 11q13 induced expression of ZNF703 mapping at 8p12, which was subsequently shown to be mediated by the Rb/E2F pathway. Nine candidate oncogenes from 8p12 and four from 11q13 were further evaluated for oncogenic function. None of the genes individually promoted colony formation in soft agar or collaborated with each other functionally. On the other hand, FGFR1 and DDHD2 at 8p12 cooperated functionally with MYC, whereas CCND1 and ZNF703 cooperated with a dominant negative form of TP53. These observations highlight the complexity and functional consequences of the genomic rearrangements that occur in these breast cancer amplicons, including transcriptional cross-talk between genes in the 8p and 11q amplicons, as well as their cooperation with major pathways of tumorigenesis.

Hedgehog signaling is often activated in tumors, yet it remains unclear how GLI2, a transcription factor activated by this pathway, acts as an oncogene. We show that GLI2 is a pleiotropic oncogene. The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2. GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2. Thus, upregulated GLI2 expression is sufficient to induce a number of the acquired characteristics of tumor cells; however, the stroma, in a tissue-specific manner, determines whether certain GLI2 oncogenic traits are expressed

Breast phyllodes tumour (PT) is a rare fibroepithelial tumour. The genetic alterations contributing to its tumorigenesis are largely unknown. To identify genomic regions involved in pathogenesis and progression of PTs we obtained genome-wide copy number profiles by array comparative genomic hybridization (CGH).DNA was isolated from fresh-frozen tissue samples. 11 PTs and 3 fibroadenomas, a frequently occurring fibroepithelial breast tumour, were analyzed. Arrays composed of 2464 genomic clones were used, providing a resolution of ~1.4 Mb across the genome. Each clone contains at least one STS for linkage to the human genome sequence.No copy number changes were detected in fibroadenomas. On the other hand, 10 of 11 PT (91%) showed DNA copy number alterations. The mean number of chromosomal events in PT was 5.5 (range 0-16) per case. A mean of 2.0 gains (range 0-10) and 3.0 losses (range 0-9) was seen per case of PT. Three cases showed amplifications. DNA copy number change was not related to PT grade. We observed recurrent loss on chromosome 1q, 4p, 10, 13q, 15q, 16, 17p, 19 and X. Recurrent copy number gain was seen on 1q, 2p, 3q, 7p, 8q, 16q, 20.In this study we used array CGH for genomic profiling of fibroepithelial breast tumours. Whereas most PT showed chromosomal instability, fibroadenomas lacked copy number changes. Some copy number aberrations had not previously been associated with PT. Several well-known cancer related genes, such as TP53 and members of the Cadherin, reside within the recurrent regions of copy number alteration. Since copy number change was found in all benign PT, genomic instability may be an early event in PT genesis.

We describe a contact printing approach for microarrays that uses fused-silica capillary tubes with tapered tips for printing pins and a pressure/vacuum system to control pin loading, printing, and cleaning. The printing process is insensitive to variable environmental factors such as humidity, and the small diameter of the pins allows routine printing from 1536 well source plates. Pin load capacity, 0.2 microL in the current system, is adjustable by controlling pin length. More than 2000 spots can be printed per 0.2-microL pin load (<100 pl/spot), and densities of >12,000 spots/cm(2) are readily achievable. Solutions with a wide range of viscosities and chemical properties can be printed. The system can print tens of thousands of different solutions at high speed, due to the ability to use large numbers of pins simultaneously, and can produce a large number of replicate arrays since all of the solution picked up by the pins is available for deposition.

SKP1-cullin-F-box protein (SCF) type ubiquitin ligases degrade proteins controlling the G1/S transition. Deficiency for FBXW7 (also known as hCDC4), which encodes the F-box protein of the SCF type ubiquitin ligase is associated with genomic instability. Here, we investigated the association of FBXW7 deficiency with chromosomal instability in breast cancer. We screened 49 tumors previously profiled by array CGH for mutations in conserved regions of FBXW7, but found no mutations. Copy number loss of FBXW7, however was associated with enhanced genomic instability in the Complex breast tumor subtype, but instability may not be due to FBXW7 haploinsufficiency, since transcript levels were not reduced in tumors with loss of the locus, whereas reduced expression was observed for other neighboring genes involved in maintenance of genome stability. We also investigated whether cells deficient for FBXW7 showed enhanced instability by challenging cells with methotrexate and assessing numbers of genomic alterations arising in resistant cells. Although methotrexate resistant colonies formed at high frequencies in HCT116 FBXW7+/- and HCT116 FBXW7-/- cells compared to parental HCT116, few copy number alterations were detected in the resistant cells. Taken together these studies suggest that FBXW7 deficiency is unlikely to contribute to the extensive copy number aberrations associated with breast and possibly other tumor types.

Human neuroblastoma remains enigmatic because it often shows spontaneous regression and aggressive growth. The prognosis of advanced stage of sporadic neuroblastomas is still poor. Here, we investigated whether genomic and molecular signatures could categorize new therapeutic risk groups in primary neuroblastomas. We conducted microarray-based comparative genomic hybridization (array-CGH) with a DNA chip carrying 2464 BAC clones to examine genomic aberrations of 236 neuroblastomas and used in-house cDNA microarrays for gene-expression profiling. Array-CGH demonstrated three major genomic groups of chromosomal aberrations: silent (GGS), partial gains and/or losses (GGP) and whole gains and/or losses (GGW), which well corresponded with the patterns of chromosome 17 abnormalities. They were further classified into subgroups with different outcomes. In 112 sporadic neuroblastomas, MYCN amplification was frequent in GGS (22%) and GGP (53%) and caused serious outcomes in patients. Sporadic tumors with a single copy of MYCN showed the 5-year cumulative survival rates of 89% in GGS, 53% in GGP and 85% in GGW. Molecular signatures also segregated patients into the favorable and unfavorable prognosis groups (P=0.001). Both univariate and multivariate analyses revealed that genomic and molecular signatures were mutually independent, powerful prognostic indicators. Thus, combined genomic and molecular signatures may categorize novel risk groups and confer new clues for allowing tailored or even individualized medicine to patients with neuroblastoma.

Tumors vary widely in chromosomal level genome instability. To gain a better understanding of the underlying defects which foster specific types of aberrations, we investigated the response of cells of related genetic backgrounds to challenge with methotrexate. We studied mismatch repair deficient HCT116 cells, two derivatives also deficient in XRCC5 (HCT116 Ku86+/-) or BLM (HCT116 BLM-/-), and mismatch repair competent HCT116+chr3 cells. We show that colony formation occurred at a significantly higher frequency in HCT116 cells and HCT116 Ku86+/- cells compared to HCT116 BLM-/- and HCT116+chr3 cells. Visible colonies arose most rapidly in HCT116 Ku86+/- cells, whereas they formed most slowly in HCT116+chr3 cells. Copy number changes acquired by the methotrexate resistant HCT116 and HCT116 BLM-/- cells most often included whole chromosome gains or losses or no acquired copy number changes, whereas resistance in HCT116+chr3 and HCT116 Ku86+/- cells was associated with amplification of DHFR and copy number transitions leading to increased copy number of DHFR, respectively. The additional copies of DHFR were present on unstable chromosomes and organized as inverted repeats in HCT116+chr3 cells, while they were most often present as direct repeats in HCT116 Ku86+/- cells. These observations suggest that different mutational mechanisms promote drug resistance in these genetic backgrounds; mismatch repair deficiency in HCT116, high rates of chromosomal instability in HCT116 Ku86+/-, and low rates of chromosomal instability in HCT116+chr3. On the other hand, it appears that loss of BLM function suppresses the mismatch repair mutator mechanism in mismatch repair and BLM deficient HCT116 BLM-/- cells.