ABSTRACT
Aberrant mitosis is a common feature of cancer, yet little is known about the altered genes causing mitotic defects. We screened human tumours for cells with morphological signatures of highly specific mitotic defects previously assigned to candidate genes in a genome-wide RNA interference screen carried out in HeLa cells (www.mitocheck.org). We discovered a striking enrichment of early mitotic configurations indicative of prophase/prometaphase delay in breast cancer. Promoter methylation analysis of MitoCheck candidate genes assigned to the corresponding 'mitotic delay' class linked this defect to epigenetic silencing of the gene encoding pregnancy-associated plasma protein-A (PAPPA), a secreted protease. PAPPA silencing was highly prevalent in precursor lesions and invasive breast cancer. Experimental manipulation of PAPPA protein levels in human mammary epithelial cells and in breast cancer cell lines demonstrates that progression through early mitosis is dependent on PAPPA function, and that breast cancer cells become more invasive after down-regulation of this protease. PAPPA regulates mitotic progression through modulating the IGF-1 signalling pathway resulting in activation of the forkhead transcription factor FoxM1, which drives a transcriptional cluster of essential mitotic genes. Our results show that PAPPA has a critical function in normal cell division and is targeted early in breast cancer development.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/physiopathology , Epigenomics , Gene Expression Regulation, Neoplastic/physiology , Gene Silencing/physiology , Mitosis/physiology , Pregnancy-Associated Plasma Protein-A/physiology , Aged , Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Progression , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Forkhead Box Protein M1 , Forkhead Transcription Factors/metabolism , Humans , Insulin-Like Growth Factor I/metabolism , Middle Aged , Phenotype , Pregnancy-Associated Plasma Protein-A/genetics , RNA Interference/physiology , Signal Transduction/physiologyABSTRACT
The mapping resolution of the physical map for chicken Chromosome 4 (GGA4) was improved by a combination of radiation hybrid (RH) mapping and bacterial artificial chromosome (BAC) mapping. The ChickRH6 hybrid panel was used to construct an RH map of GGA4. Eleven microsatellites known to be located on GGA4 were included as anchors to the genetic linkage map for this chromosome. Based on the known conserved synteny between GGA4 and human Chromosomes 4 and X, sequences were identified for the orthologous chicken genes from these human chromosomes by BLAST analysis. These sequences were subsequently used for the development of STS markers to be typed on the RH panel. Using a logarithm of the odds (LOD) threshold of 5.0, nine linkage groups could be constructed which were aligned with the genetic linkage map of this chromosome. The resulting RH map consisted of the 11 microsatellite markers and 50 genes. To further increase the number of genes on the map and to provide additional anchor points for the physical BAC map of this chromosome, BAC clones were identified for 22 microsatellites and 99 genes. The combined RH and BAC mapping approach resulted in the mapping of 61 genes on GGA4 increasing the resolution of the chicken-human comparative map for this chromosome. This enhanced comparative mapping resolution enabled the identification of multiple rearrangements between GGA4 and human Chromosomes 4q and Xp.
