Cell invasion through the basement membrane is a key mechanism underlying cell dispersal and organ formation during normal development, immune surveillance and is dysregulated during cancer metastasis. Yet due to dif?culties of studying these dynamic behaviors in vivo, it is the least understood aspect of the metastatic cascade. My laboratory utilizes a powerful in vivo model to examine cell invasive behavior, by combining functional genomic and genetic tools with single-cell visual analyses. We examine anchor cell (AC) invasion into the vulval epithelium during C. elegans larval development. Data from our laboratory has shown a functional link between G1 phase cell cycle arrest and the acquisition of an invasive phenotype. Through high- throughput screens, we have identi?ed that the activity of a single conserved NR2E1 class nuclear hormone receptor, the transcription factor, nhr-67 (TLX), is required to maintain the invasive AC in G1 cell cycle arrest. Loss of nhr-67 results in mitotic ACs that fail to invade. Strikingly, AC invasion can be rescued by preventing cell division through induction of G1 cell cycle arrest. Downstream of G1 arrest, chromatin modi?ers, including the histone deacetylase, hda-1, are required for expression of pro-invasive genes, including matrix metalloproteinases (MMPs) and regulators of the F-actin cytoskeleton, leading to differentiation of the invasive phenotype. For this proposal we plan to elucidate how cell cycle arrest is functionally linked to invasion.
In Aim 1, using molecular epistatic interaction experiments and inducible transgene expression, we will identify the upstream network of transcription factors that mediate NHR-67 activity.
In Aim 2, we will generate CRISPR/Cas9-mediated conditional alleles of cell cycle control genes to determine how the AC maintains G1 arrest.
In Aim 3, we will pair tissue-speci?c RNAi screening of chromatin modi?ers with high resolution confocal and structured illumination (SIM) imaging of sub-nuclear organization, to understand the link between invasion and differentiation. The results of these aims will provide the ?rst mechanistic view of the transcriptional and epigenetic control of cell cycle arrest and invasive behavior. Thus, our proposed work has the potential to provide a mechanistic understanding of how cells acquire and maintain an invasive phenotype, a critical aspect of many developmental and genetic disorders.
/Public Health Relevance Statement The acquisition of cell invasive behavior is critical to trophoblast implantation, cardiac development, neural crest migration and leukocyte transmigration. Dysregulation of invasive activity is the fundamental step in dissemination of tumor cells during metastasis. Determination of the mechanisms that mediate how cell cycle regulation directly controls the ability for cells to adopt an invasive phenotype will increase our understanding of diseases that stem from inappropriate invasive pathologies, including pre-eclampsia, asthma, rheumatoid arthritis and cancer. Our results will hopefully lead to improved design of therapeutics to modulate invasive behavior during disease progression.
|Liu, Tsung-Li; Upadhyayula, Srigokul; Milkie, Daniel E et al. (2018) Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science 360:|
|Medwig, Taylor N; Matus, David Q (2017) Breaking down barriers: the evolution of cell invasion. Curr Opin Genet Dev 47:33-40|