Abstract
The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2−/− MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2−/− MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.
Highlights
SALL2 is a member of the Spalt/Sal family of transcription factors characterized by their role in organ development and conserved from Caenorhabditis elegans to humans.[5,6,7]
We show that Sall[2] is required for full apoptotic response to doxorubicin, and demonstrate that apoptosis is significantly decreased in Sall2-deficient cells even in the presence of activated p53
We demonstrate that Sall[2] binds to and transactivates the NOXA promoter under genotoxic stress in vitro and in vivo
Summary
SALL2 is a member of the Spalt/Sal family of transcription factors characterized by their role in organ development and conserved from Caenorhabditis elegans to humans.[5,6,7] Sall2deficient mice were previously reported to have no apparent abnormal phenotype when bred on a C57BL/6 genetic background; a strain-specific incidence of neural tube defects and perinatal lethality were reported when bred on mixed genetic backgrounds.[8,9] Recently, it was demonstrated that Sall[2] has a role in eye morphogenesis and a SALL2 gene mutation was associated with coloboma, a congenital eye defect.[2,10] A deleterious SALL2 mutation was associated with Alport syndrome, a renal disease,[1] suggesting that Sall[2] could have a role in kidney development. A novel Sall[2] target during genotoxic stress D Escobar et al factor), the proapoptotic gene BAX (B-cell lymphoma 2 (BCL2)-associated X protein) and the proto-oncogene c-Myc. Sall[2] upregulates p21WAF and BAX, it represses c-Myc.[12,20,21] Sall[2] targets were identified in a p53-independent context, with Sall[2] and p53 having common targets. We show that the Sall2/Noxa axis is important for the cell death response to doxorubicin in Jurkat T cells, supporting the significance of this axis in a cancer cell context This new data supports the tumor suppressor function of Sall[2], and enhances the understanding of Sall[2] role in the context of genotoxic stress
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