Computational identification of a p38SAPK-regulated transcription factor network required for tumor cell quiescence

Abstract

The stress-activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools, we identified a transcription factor (TF) network regulated by p38alpha/beta and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Furthermore, induction of p53 by p38 was dependent on c-Jun down-regulation. Accordingly, RNAi down-regulation of BHLHB3 or p53 interrupted tumor cell quiescence, while down-regulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers.

Figure 1

A Venn diagrams of genes changing significantly (91 up, 133 down) to each of the three strategies using both the gcRMA and MAS5 methods. Significance was established with a p value of the F-statistic at 0.05. Total number of genes induced or repressed by p38 are indicated outside the Venn diagram. B, TF network regulated by p38 signaling showing only genes that move significantly up or down in all three of the p38 inhibition experiments and the TFs that potentially control them. TFs are depicted as boxes and target genes other than TFs with circles. The colors indicate whether they were upregulated (red) or downregulated (green) by p38 signaling. Where a gene's promoter contains known/predicted binding sites for a given TF, the binding sites scores are summed and multiplied by their expression correlation (Spearman across all samples). An arrow is then drawn between the TF and gene where the score exceeds a threshold of 0.75. Arrows colors indicate whether the genes were negatively (orange) or positively (green) co-regulated. White boxes depict changes in TF expression that were not statistically significant but still were significantly correlated with the putative target genes. The expression level trend of white box TFs can be inferred by the color of the connecting arrow.

Figure 2

BHLHB3 is required for D-HEp3 cell quiescence A, basal expression of BHLHB3 detected by RT-PCR (left panel) or (right panel) qPCR under the indicated treatments for 24hrs. PD = 20 µM PD98059 Mek1/2 inhibitor, SB=10 µM SB203580 p38 inhibitor; DMSO= dimethyl sulfoxide used as vehicle. B, siRNA-mediated knockdown of BHLHB3 or p38α but not p53, caused a strong decrease in BHLHB3 mRNA as measured by RT-PCR (upper panel) or qPCR (middle panel) and interrupts the quiescence of D-HEp3 cells (lower panel). C, shRNA stable knockdown of BHLHB3 measured by RT-PCR (left panel) not observed with shRNAs to luciferase (Luc) results in restored proliferation of D-HEp3 cells in vivo (right panel). Lower panel, when inoculated in nude mice (2×10⁵ cells/mouse), D-HEp3 cells expressing BHLHB3 shRNA have a statistically significant shortening in their dormancy period. D, Overexpression of V5-BHLHB3 inhibits proliferation of T-HEp3 cells on CAMs in vivo (left panel). Right panel, detection of the V5-tag after transfection of T-HEp3 cells with an empty vector or V5-BHLHB3. MKK6 is not tagged and its expression has been verified (21).

Figure 3

A, Basal c-Jun expression is lower in D-HEp3 cells than in T-HEp3 cells, increased by 10 µM SB203580 (SB) treatment in D-HEp3 cells and reduced by 20 µM PD98059 (PD) in T-HEp3 cells as measured by RT-PCR (upper panel) and qPCR (middle panel).B, SiRNA mediated down-regulation of c-Jun detected by Western blot (upper panel) reduced cell proliferation of T-HEp3 cells after 4 days in vivo (lower panel). Scrambled siRNAs served as controls. C, Quantification of apoptosis (cleaved caspase-3 staining) in 2 day old tumors of T-HEp3 cells transfected with control, c-Jun or FoxM1 siRNAs (left panel). Right panel: quantification of phospho-histone H3 levels in the same samples (triplicate experiments). D, Basal (DMSO) FoxM1 expression level is lower in D-HEp3 cells than in T-HEp3 cells and can be induced by 10µM SB203580 (SB) treatment in D-HEp3 cells (upper panel). siRNA-mediated knockdown of FoxM1 for 48 hrs, as detected by qPCR (lower left panel), reduced cell expansion of T-HEp3 cells after 3 days in vivo (lower right panel).

Figure 4

A, protein sequence alignment along aa 201–250 between wt (NM_000546) and mutant p53 in HEp3 cells shows an R213Q substitution (upper panel). Basal p53 mRNA (middle panel) and protein (lower panel) expression is higher in D-HEp3 cells than in T-HEp3 cells; the lanes show treatments with vehicle (DMSO) or Mek1/2 inhibitor PD98059 (20µM) or p38 inhibitor SB203580 (10 µM), as measured by RT-PCR and Western blot, respectively. B, p53 luciferase reporter activity measured in vivo was higher in D-HEp3 cells than T-HEp3 cells after 72h in vivo growth on CAMs. C, shRNA knockdown of p53 measured by Western blot (upper panel), results in restored proliferation of D-HEp3 cells in vivo (lower panel). D, Knockdown of p53 by siRNAs in DHEp3 cells was measured at the protein level by Western blot and qPCR (upper panel), promoted cell proliferation in vivo in D-HEp3 cells (lower panel). All experiments performed at least in triplicate.