Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin

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.

mRNA and methylation profiling of radioresistant esophageal cancer cells: the involvement of Sall2 in acquired aggressive phenotypes

“…Sall2 (Sal-like protein 2), a homeotic transcription factor, is commonly considered as a tumor suppressor 41, 42. Cells with deletion of Sall2 ( Sall2 -/- MEFs) showed reduced apoptosis with increased cell viability in response to genotoxic stress 43. Consistent with this report, in our study, we found decreased Sall2 expression in radioresistant cells with reduced cell death in response to radiation and cisplatin.…”

“…To further investigate the induced function of Sall2, we overexpressed Sall2 and found reversed malignancy of these cancer cells, indicating that Sall2 plays a role in radioresistance. A recent study shows that Sall2 positively regulates Noxa and involves in the apoptotic response to extended genotoxic stress in cancer cells 43. As a transcriptional factor, Sall2 is likely to activate or repress its target genes.…”

SALL2 represses cyclins D1 and E1 expression and restrains G1/S cell cycle transition and cancer‐related phenotypes

“…The SV40 large T antigen expression pBSSVD2005 plasmid was a gift from David Ron (Addgene plasmid # 21826), the plasmid containing the CCNE1 promoter was a gift from Bob Weinberg (Addgene plasmid # 8458) (Geng et al ., ), and the CCND1 promoter pGL3Basic was a gift from Frank McCormick (Addgene plasmid # 32726) (McCormick and Tetsu, ). pcDNA3‐SALL2 plasmid was described elsewhere (Escobar et al ., ). Alexa Fluor 488‐conjugated phalloidin and Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).…”

“…Sall2 +/+ and Sall2 −/− fibroblasts were prepared from embryos at 13.5 days postcoitum as previously described (Escobar et al ., ). Briefly, embryos, whose head and other red organs were removed, were smashed into pieces using a razor blade in a 100‐mm dish with 5 mL trypsin (GE Healthcare HyClone, Logan, UT, USA).…”

“…SALL2 has also been associated with induction of cellular quiescence in human fibroblasts (Liu et al ., ) and with cellular apoptosis in mouse embryonic fibroblast and human leukemia cells exposed to genotoxic stress (Escobar et al ., ). Clinical evidence showed loss of heterozygosity (LOH) at the SALL2 locus in 30% of ovarian cancer patients (Bandera et al ., ), and recent studies demonstrated that the P2 promoter of SALL2 is susceptible to silencing by methylation (Sung et al ., ).…”