Identification of putative cancer genes through data integration and comparative genomics between plants and humans

Abstract

Coordination of cell division with growth and development is essential for the survival of organisms. Mistakes made during replication of genetic material can result in cell death, growth defects, or cancer. Because of the essential role of the molecular machinery that controls DNA replication and mitosis during development, its high degree of conservation among organisms is not surprising. Mammalian cell cycle genes have orthologues in plants, and vice versa. However, besides the many known and characterized proliferation genes, still undiscovered regulatory genes are expected to exist with conserved functions in plants and humans. Starting from genome-wide Arabidopsis thaliana microarray data, an integrative strategy based on coexpression, functional enrichment analysis, and cis-regulatory element annotation was combined with a comparative genomics approach between plants and humans to detect conserved cell cycle genes involved in DNA replication and/or DNA repair. With this systemic strategy, a set of 339 genes was identified as potentially conserved proliferation genes. Experimental analysis confirmed that 20 out of 40 selected genes had an impact on plant cell proliferation; likewise, an evolutionarily conserved role in cell division was corroborated for two human orthologues. Moreover, association analysis integrating Homo sapiens gene expression data with clinical information revealed that, for 45 genes, altered transcript levels and relapse risk clearly correlated. Our results illustrate how a systematic exploration of the A. thaliana genome can contribute to the experimental identification of new cell cycle regulators that might represent novel oncogenes or/and tumor suppressors.

Fig. 1

Schematic representation of the applied methodology for the selection of the target genes using data integration and comparative genomics. Starting from genome-wide Arabidopsis thaliana microarray data, an integrative strategy based on coexpression, functional enrichment analysis, and cis-regulatory element annotation was combined with a comparative genomics approach between plants and humans to detect conserved cell cycle genes involved in DNA replication and DNA repair processes. Numbers in parentheses report the number of genes that were retained after each step

Fig. 2

Experimental association of Arabidopsis thaliana candidate genes with cell replication. The Arabidopsis first leaf was used to measure cell division and DNA replication parameters. a The mean number of endoreduplication cycles denoted as Endoreduplication Index (EI) of the T-DNA insertion lines [*statistically different from the control (Col-0) plants, according to the t test P < 0.05 (n = 10); ± represents hemizygous mutants]. b Scatter plot of the analyzed mutants. Mutants were plotted according to their respective number of cells and cell size. Mutant lines are color-coded according to their DNA ploidy content phenotype. Green and red dots represent mutants with a reduced and increased EI, respectively

Fig. 3

Hypersensitivity of selected T-DNA insertion lines towards DNA replication-inhibiting treatments. a Root length under standard growth conditions for the analyzed T-DNA insertion lines. Roots were measured after 7 days of growth on vertical MS plates. b, c Mutants displaying a differential root growth response upon UV-B irradiation or in the presence of 1 mM HU, respectively [*Statistically different from the control (Col-0) plants according to the t test P < 0.05 (n = 30); ± represents hemizygous mutants]

Fig. 4

Experimental association of human candidate genes with cell division in MCF7 cell cultures. Genes were silenced in breast epithelial cancer cell cultures (MCF7 cells) using small interfering (si) RNA sequences. a Growth curves of the siRNA knocked-down MCF7 cultures, illustrating growth inhibition by knock-down of C14ORF21 and HEATR6. b Ploidy distributions of the STATIP1, C14ORF21, and HEATR6 knocked-down cultures in comparison with controls assessed by flow cytometry, illustrating a significant increased number of G2/M cells in C14ORF21 and HEATR6 knock-down cultures (P < 0.05 (n = 9) according to a t test). c Expression levels of cell cycle phase makers measured by Q-PCR, illustrating transcriptional upregulation of the G2/M marker genes CDK1, CyclinB1, and CyclinB2 in C14ORF21 and HEATR6 knock-down cell cultures, indicative for a transient G2 arrest

Fig. 5

Association of human orthologues of Arabidopsis genes involved in cell replication with specific cancer outcomes (relapse risk). Cox survival plots for the human orthologues of Arabidopsis genes with a direct influence on cell proliferation were constructed. A clear association between increased gene expression levels and a diminished probability of relapse-free survival is shown. RRC relative risk coefficient, *statistically significant differences in the survival probability, P < 0.01