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Jiang Cheng Shen1, Kensuke Kumamoto1, Motoko Unoki1, Lyuba Varticovski1, Remy Pedeux2, and Curtis C. Harris1 1Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA, and 2INSERM U578, Institut Albert Bonniot, France The candidate tumor suppressor ING4 belongs to a gene family initially manifested in inhibition of cell growth involving in p53-dependent gene regulation pathways. Recent studies further suggested the ING4 function in regulation of angiogenesis induced by human glioblastoma cells through the NF-κB pathway; and in suppression of lost of contact inhibition by ectopic expression of human ING4 in myc-elicited rat cells. Thus ING4 may play a role in modulation of cell morphology and/or cell motility. Here we report that ING4 regulates cell spreading and migration. We first demonstrated that expression of ING4 in RKO cells suppresses cell migration in modified Boyden chamber and siRNA knock-down of ING4 enhances the migration. Observation of single cell spreading by phalloidin staining on F-actin also indicated that ING4 modulates actin filament organization and thus slows down cell spreading, which is consistent with results from monolayer wound-healing assay that ING4 expression in RKO cells retards the gap-filling capability after inflicting a scratch wound on the cell monolayer. Cytoplasmic localization of ING4 can be visualized by immunofluorescence microscopy and ING4 proteins are accumulating in the protruding lamellipodium, suggesting a role for ING4 in cytoskeleton organization during cell spreading. To understand the network of ING4-interacting proteins, we carried out protein affinity pull-down and by using mass spectrometry we identified a target protein, liprin α1, which plays an important role in regulation of focal adhesion. Subsequent co-immunoprecipitation of ING4 and liprin α1 further confirmed this physical interaction, by which we suggest a possible role of ING4 in focal adhesion regulation. Further analyses by immunofluorescence showed that ING4 either co-localizes with, or proximately localizes to, the components of focal adhesion complex during cell spreading. We thus propose that ING4 involves in regulation of focal adhesion during cell spreading and migration. In summary, we have demonstrated that ING4 regulates cell spreading and migration. The cytoplasmic functions of ING4, including accumulation in lamellipodium, co-localization with focal adhesion complex, and interaction with liprin α1, have provided molecular evidence to substantiate this observation. Our results therefore shed a new light on studies for ING4 function in signal transduction and tumor suppression.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]