The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time...[ Read more ]

The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects caused by hIPI3 silencing. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

PCM1 (Pericentriolar Material-1) is a component of centriolar satellites and acts as a scaffold to target several proteins to the centrosome in a dynein motor-dependent manner and to regulate microtubule dynamics. PCM1 is also important for the formation of primary cilia by associating with CEP290 and the BBSome during ciliogenesis. Emerging evidence suggests that cilia also play key roles in tumorigenesis and/or cancer development. Furthermore, our exome sequencing data suggested that ciliogenesis genes may be involved in lung cancer metastasis. Therefore, the aim of this part of my study is to understand the role of PCM1 in lung cancer cell migration.

The results of our wound-healing assay and trans-well invasion assay showed that knockdown of PCM1 strongly inhibited cell migration and invasion of A549 lung cancer cells. On the other hand, knockdown of PCM1 had no effect on cell proliferation, which was demonstrated by cell viability assay and colony formation assay. Moreover, the percentage of cells with cilia was significantly reduced in A549 cells after PCM1 silencing. Interesting, during cell migration, more than 50% of cells in the first row of the migrating edge had cilia in cells without PCM1 knockdown, whereas only about 20% in PCM1 silenced cells. Taken together, these data suggest that PCM1 and cilia play a critical role in lung cancer cell migration and invasion.