The online version of this article (doi:10.1186/1476-4598-11-46) contains supplementary material, which is available to authorized users.
Toshiyuki Tsunoda, Takeharu Ota contributed equally to this work.
The authors declare that they have no competing interests.
TT performed the statistical analysis, coordinated the experiments, and drafted the manuscript. TO carried out the cell and molecular biology experiments and drafted the manuscript. TF, YT, YY, MO and HM performed the cellular assays, molecular studies, western blots and RT-PCR. KD carried out the microarray gene expression analysis and bioinformatics. DRT contributed interpretation and analyzed data. MK and SM participated in study design. SS contributed to data evaluation, interpretation and drafted the manuscript. All authors have read and approved the final manuscript.
We previously established a three-dimensional (3-D) colonic crypt model using HKe3 cells which are human colorectal cancer (CRC) HCT116 cells with a disruption in oncogenic KRAS, and revealed the crucial roles of oncogenic KRAS both in inhibition of apoptosis and in disruption of cell polarity; however, the molecular mechanism of KRAS-induced these 3-D specific biological changes remains to be elucidated.
Among the genes that were upregulated by oncogenic KRAS in this model, we focused on the phosphodiesterase 4B (PDE4B) of which expression levels were found to be higher in clinical tumor samples from CRC patients in comparison to those from healthy control in the public datasets of gene expression analysis. PDE4B2 was specifically overexpressed among other PDE4 isoforms, and re-expression of oncogenic KRAS in HKe3 cells resulted in PDE4B overexpression. Furthermore, the inhibition of PDE4 catalytic activity using rolipram reverted the disorganization of HCT116 cells into the normal physiologic state of the epithelial cell polarity by inducing the apical assembly of ZO-1 (a tight junction marker) and E-cadherin (an adherens junction marker) and by increasing the activity of caspase-3 (an apoptosis marker) in luminal cavities. Notably, rolipram reduced the AKT phosphorylation, which is known to be associated with the disruption of luminal cavity formation and CRC development. Similar results were also obtained using PDE4B2-shRNAs. In addition, increased expression of PDE4B mRNA was found to be correlated with relapsed CRC in a public datasets of gene expression analysis.
These results collectively suggested that PDE4B is upregulated by oncogenic KRAS, and also that the inhibition of PDE4 catalytic activity can induce both epithelial cell polarity and luminal apoptosis in CRC, thus highlighting the utility of our 3-D culture (3 DC) model for the KRAS-induced development of CRC in 3-D microenvironment. Indeed, using this model, we found that PDE4B is a promising candidate for a therapeutic target as well as prognostic molecular marker in CRC. Further elucidation of the signaling network of PDE4B2 in 3 DC would provide a better understanding of CRC in vivo.
Additional file 1: Additional Table 1. Upregulated genes by oncogenic KRAS in 3 DC (HCT116/HKe3 in 3 DC; Score > 10.00; 387 genes). (XLS 69 KB)12943_2011_1017_MOESM1_ESM.xls
Additional file 2: Additional Table 2. List of the forward and reverse primers used to amplify PDE4 variants and β-actin. (XLS 26 KB)12943_2011_1017_MOESM2_ESM.xls
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- Inhibition of Phosphodiesterase-4 (PDE4) activity triggers luminal apoptosis and AKT dephosphorylation in a 3-D colonic-crypt model
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