BioProject

Purpose: The goal of this study was to determine a specific gene expression profile of Nuclear Aurora-A Kinase expressing cells. It has been previously shown that AURKA localizes to the nucleus in patient biopsies in multiple aggressive malignancies including breast cancer but functional significance of this localization in cancer progression is still undetermined. To define the role nuclear AURKA plays in metastatic dissemination and colonization of distant organs, we created TNBC (triple negative breast cancer) cell lines with CRISPR/Cas9 based deletion of endogenous AURKA and rescue with exogenous AURKA specifically targeted to the nucleus, cytoplasm or both. The TNBC cells expressing nuclear AURKA show significant increase in dissemination and colonization of multiple organs when compared to controls. The RNA sequencing analysis of nuclear AURKA expressing cell lines shows significant changes in gene expression profile associated with hypoxia, migration/invasion and survival/death pathways supporting the metastatic phenotype observed in vivo. Methods: The total RNA was isolated using Quagen RNeasy kit, the library was build using KAPA mRNA Hyper Prep Kit with TruSeq adapters for the build, RNA profiles of WT-AURKA, NES-AURKA, NLS-AURKA and Empty (including no-localization tag-Empty, NLS-empty, NES-empty) vector controls expressing MDA-MB-231 cells were generated by deep sequencing of 2 biological clones of each cell line in triplicate, using Illumina HiSeq2500, 2 x 50 paired-end reads in the High Output Mode. The sequence reads that passed quality filters were analyzed with Hisat2 followed by Samtools. Genes whose expression is changed between the control samples and WT, NES, NLS-AURKA samples were identified using Partek Genomic Suite and functions associated with that set of genes mapped using Ingenuity Pathway Analysis. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we identified 33,563 transcripts after mapping reads to the human genome. Approximately 3,000 of the transcripts showed differential expression between the Empty control and NLS-AURKA and NES-AURKA, with a fold change ≥1.5 and p value <0.05. Altered expression of 96 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several well characterized genes that contribute to hypoxia, migration/invasion and survival/death pathways. Data analysis revealed a significant changes in transcriptome profiling upon introduction of AURKA in the nucleus supporting its active role on regulation of transcription. Conclusions: Our study represents the first detailed analysis of transcriptome changes upon manipulation of AURKA localization in triple negative breast cancer cells, with multiple biologic and technical replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles in patient samples in future studies. Our results show that AURKA expression in nucleus might be a key event triggering metastatic dissemination and offers a comprehensive and qualitative evaluation of mRNA content. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions of nuclear AURKA. Overall design: The mRNA profiles of MDA-MB-231 cells with variable subcellular localization of Aurora-A Kinase in Cytoplasm or Nucleus or both were generated by deep sequencing, in triplicate, using Illumina HiSeq and correlated with metstatic potenital of these cell in vivo xenograft studes.