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A hidden role of the inactivated FANCD2: upregulating ΔNp63.

Panneerselvam J, Pickering A, Zhang J, Wang H, Tian H, Zheng J, Fei P - Oncotarget (2013)

Bottom Line: Here we unexpectedly found that ΔNp63 mRNA was expressed at high levels in human cancer cells carrying an impaired FA pathway compared to the corresponding control cells carrying an intact FA pathway.In vivo, the elevation of FAVL, a tumor promotion factor that inhibits FANCD2 activation, was found to be positively associated with ΔNp63 expression in human cancer tissues.Collectively, these results document a novel role of an inactivated FANCD2 in upregulating ΔNp63, advancing our understanding of how an impaired FA pathway contributes to the pathogenesis of human cancer.

View Article: PubMed Central - PubMed

Affiliation: University of Hawaii Cancer Center, University of Hawaii, Honolulu, HI, USA.

ABSTRACT
A compromised Fanconi Anemia (FA) signaling pathway, often resulting from an inactivated FANCD2, was recently recognized to contribute to the development of non-FA human tumors. However, it is largely unknown as to how an impaired FA pathway or an inactivated FANCD2 promotes tumorigenesis. Here we unexpectedly found that ΔNp63 mRNA was expressed at high levels in human cancer cells carrying an impaired FA pathway compared to the corresponding control cells carrying an intact FA pathway. This observation was recapitulated upon conditionally managing the status of FANCD2 monoubiquitination /activation in 293T cells. Importantly, ΔNp63 elevation upon FANCD2 inactivation was confirmed in human fibroblasts derived from FA patients. Moreover, we have identified a 189 bp DNA fragment downstream of the ΔNp63 promoter (P2) that can mediate the upregulation of ΔNp63 by an inactivated FANCD2, and determined that elevated ΔNp63 is high enough to promote cancer cell proliferation and metastasis. In vivo, the elevation of FAVL, a tumor promotion factor that inhibits FANCD2 activation, was found to be positively associated with ΔNp63 expression in human cancer tissues. Collectively, these results document a novel role of an inactivated FANCD2 in upregulating ΔNp63, advancing our understanding of how an impaired FA pathway contributes to the pathogenesis of human cancer.

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Inactivated FANCD2 promotes the expression of ΔNp63 via a 1.2 kb DNA fragment downstream of the P2 promoter(A) Schematic representation of 1 kb and 1.2 kb DNA fragments up or downstream of the P2 promoter. (B) Inactivated FANCD2 enhances the 1.2 kb reporter activity. Both the 1.0 kb and 1.2 kb DNA fragments (Fig. 3A) were individually cloned into the upstream of pGL-3-promoter- reporter, named 1 kb or 1.2 kb reporters. Cells showed a higher reporter activity when the 1.2kb reporter was co-transfected with K561R FANCD2 cDNA-containing plasmid. The relative reporter activity was plotted upon photon counts as we did previously. Cells carrying the 1.0kb reporter along with either wt or mtFANCD2 cDNA did not show a noticeable difference in the reporter activity as compared to the 1.0 kb reporter alone (not shown). The results shown are a representative of five independent experiments performed each time in triplicate, and error bars indicate the standard deviation. The transfection efficiency of the reporter assay for pEGFP-Flag-wtFANCD2 or -mtFANCD2 was measured via western blotting analysis with antibodies against GFP (the pEGFP vector produces polycistronic mRNAs encoding non-fusion GFP protein) and Flag-fused FANCD2 protein. (C) The inactivated FANCD2 associates more strongly with the 1.2 kb DNA fragment. Both HCT116 and U2OS sets of stably-transfected cell pairs carrying an intact or impaired FA pathway, respectively (Supplementary Figures 1 and 3 right panel) were used to perform FANCD2 ChIP analysis using primers to bracket DNA fragments 1 kb up or 1.2 kb downstream of the P2 promoter (Figure 3A). (The relative folds were calculated upon the band density measured by NIH image J program with the corresponding bands generated from control cells as “1”.)
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Figure 3: Inactivated FANCD2 promotes the expression of ΔNp63 via a 1.2 kb DNA fragment downstream of the P2 promoter(A) Schematic representation of 1 kb and 1.2 kb DNA fragments up or downstream of the P2 promoter. (B) Inactivated FANCD2 enhances the 1.2 kb reporter activity. Both the 1.0 kb and 1.2 kb DNA fragments (Fig. 3A) were individually cloned into the upstream of pGL-3-promoter- reporter, named 1 kb or 1.2 kb reporters. Cells showed a higher reporter activity when the 1.2kb reporter was co-transfected with K561R FANCD2 cDNA-containing plasmid. The relative reporter activity was plotted upon photon counts as we did previously. Cells carrying the 1.0kb reporter along with either wt or mtFANCD2 cDNA did not show a noticeable difference in the reporter activity as compared to the 1.0 kb reporter alone (not shown). The results shown are a representative of five independent experiments performed each time in triplicate, and error bars indicate the standard deviation. The transfection efficiency of the reporter assay for pEGFP-Flag-wtFANCD2 or -mtFANCD2 was measured via western blotting analysis with antibodies against GFP (the pEGFP vector produces polycistronic mRNAs encoding non-fusion GFP protein) and Flag-fused FANCD2 protein. (C) The inactivated FANCD2 associates more strongly with the 1.2 kb DNA fragment. Both HCT116 and U2OS sets of stably-transfected cell pairs carrying an intact or impaired FA pathway, respectively (Supplementary Figures 1 and 3 right panel) were used to perform FANCD2 ChIP analysis using primers to bracket DNA fragments 1 kb up or 1.2 kb downstream of the P2 promoter (Figure 3A). (The relative folds were calculated upon the band density measured by NIH image J program with the corresponding bands generated from control cells as “1”.)

Mentions: To define the association between inactivated FANCD2 and an enhanced level of ΔNp63 expression, we asked whether inactivated FANCD2 plays a direct role in the regulation of ΔNp63 mRNA expression. We constructed a ΔNp63 promoter (P2)-containing luciferase reporter, and co-transfected the reporter with wt or K561R FANCD2 cDNA-containing plasmids (K561R FANCD2 cDNA encodes a FANCD2 protein lacking the lysine residue required for monoubiquitination). We did not observe any change in reporter activity (data not shown) to support the above finding (Figures 1 and 2). We also performed chromatin immunoprecipitation (ChIP), which did not show an interaction between the P2 promoter and mtFANCD2 nor wtFANCD2 (data not shown). Considering enhancers that can promote transcription, we used the sequences 1 kb upstream and 1.2 kb downstream of the P2 promoter to construct two new reporters respectively (Figure 3A). Through the reporter assay, we found that cells carrying mtFANCD(K561R)-containing plasmid showed a higher luciferase activity when co-transfected with the reporter containing the 1.2 kb DNA fragment downstream of P2 as compared to the cells transfected with empty vector or wtFANCD2 in various combinations, all of which showed a similar basal level of luciferase activity (Figure 3B; data not shown). These results suggest that inactivated FANCD2 may play a role in enhancing the transcription of ΔNp63, which appears to be a new function for the inactivated FANCD2, rather than a loss function of wtFANCD2. To support the reporter activity observed, we conducted ChIP analysis on the binding potential of inactivated FANCD2 protein to the 1.2 kb DNA fragment. We found that antibodies against inactivated FANCD2 can also pull down a substantial amount of the downstream DNA fragment, but not the one upstream of the P2 promoter (Figure 3C), which agrees with the reporter assay (Figure 3B). Therefore, inactivated FANCD2 can regulate ΔNp63 mRNA expression through the association with a DNA sequence downstream of the known P2 promoter of ΔNp63.


A hidden role of the inactivated FANCD2: upregulating ΔNp63.

Panneerselvam J, Pickering A, Zhang J, Wang H, Tian H, Zheng J, Fei P - Oncotarget (2013)

Inactivated FANCD2 promotes the expression of ΔNp63 via a 1.2 kb DNA fragment downstream of the P2 promoter(A) Schematic representation of 1 kb and 1.2 kb DNA fragments up or downstream of the P2 promoter. (B) Inactivated FANCD2 enhances the 1.2 kb reporter activity. Both the 1.0 kb and 1.2 kb DNA fragments (Fig. 3A) were individually cloned into the upstream of pGL-3-promoter- reporter, named 1 kb or 1.2 kb reporters. Cells showed a higher reporter activity when the 1.2kb reporter was co-transfected with K561R FANCD2 cDNA-containing plasmid. The relative reporter activity was plotted upon photon counts as we did previously. Cells carrying the 1.0kb reporter along with either wt or mtFANCD2 cDNA did not show a noticeable difference in the reporter activity as compared to the 1.0 kb reporter alone (not shown). The results shown are a representative of five independent experiments performed each time in triplicate, and error bars indicate the standard deviation. The transfection efficiency of the reporter assay for pEGFP-Flag-wtFANCD2 or -mtFANCD2 was measured via western blotting analysis with antibodies against GFP (the pEGFP vector produces polycistronic mRNAs encoding non-fusion GFP protein) and Flag-fused FANCD2 protein. (C) The inactivated FANCD2 associates more strongly with the 1.2 kb DNA fragment. Both HCT116 and U2OS sets of stably-transfected cell pairs carrying an intact or impaired FA pathway, respectively (Supplementary Figures 1 and 3 right panel) were used to perform FANCD2 ChIP analysis using primers to bracket DNA fragments 1 kb up or 1.2 kb downstream of the P2 promoter (Figure 3A). (The relative folds were calculated upon the band density measured by NIH image J program with the corresponding bands generated from control cells as “1”.)
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Figure 3: Inactivated FANCD2 promotes the expression of ΔNp63 via a 1.2 kb DNA fragment downstream of the P2 promoter(A) Schematic representation of 1 kb and 1.2 kb DNA fragments up or downstream of the P2 promoter. (B) Inactivated FANCD2 enhances the 1.2 kb reporter activity. Both the 1.0 kb and 1.2 kb DNA fragments (Fig. 3A) were individually cloned into the upstream of pGL-3-promoter- reporter, named 1 kb or 1.2 kb reporters. Cells showed a higher reporter activity when the 1.2kb reporter was co-transfected with K561R FANCD2 cDNA-containing plasmid. The relative reporter activity was plotted upon photon counts as we did previously. Cells carrying the 1.0kb reporter along with either wt or mtFANCD2 cDNA did not show a noticeable difference in the reporter activity as compared to the 1.0 kb reporter alone (not shown). The results shown are a representative of five independent experiments performed each time in triplicate, and error bars indicate the standard deviation. The transfection efficiency of the reporter assay for pEGFP-Flag-wtFANCD2 or -mtFANCD2 was measured via western blotting analysis with antibodies against GFP (the pEGFP vector produces polycistronic mRNAs encoding non-fusion GFP protein) and Flag-fused FANCD2 protein. (C) The inactivated FANCD2 associates more strongly with the 1.2 kb DNA fragment. Both HCT116 and U2OS sets of stably-transfected cell pairs carrying an intact or impaired FA pathway, respectively (Supplementary Figures 1 and 3 right panel) were used to perform FANCD2 ChIP analysis using primers to bracket DNA fragments 1 kb up or 1.2 kb downstream of the P2 promoter (Figure 3A). (The relative folds were calculated upon the band density measured by NIH image J program with the corresponding bands generated from control cells as “1”.)
Mentions: To define the association between inactivated FANCD2 and an enhanced level of ΔNp63 expression, we asked whether inactivated FANCD2 plays a direct role in the regulation of ΔNp63 mRNA expression. We constructed a ΔNp63 promoter (P2)-containing luciferase reporter, and co-transfected the reporter with wt or K561R FANCD2 cDNA-containing plasmids (K561R FANCD2 cDNA encodes a FANCD2 protein lacking the lysine residue required for monoubiquitination). We did not observe any change in reporter activity (data not shown) to support the above finding (Figures 1 and 2). We also performed chromatin immunoprecipitation (ChIP), which did not show an interaction between the P2 promoter and mtFANCD2 nor wtFANCD2 (data not shown). Considering enhancers that can promote transcription, we used the sequences 1 kb upstream and 1.2 kb downstream of the P2 promoter to construct two new reporters respectively (Figure 3A). Through the reporter assay, we found that cells carrying mtFANCD(K561R)-containing plasmid showed a higher luciferase activity when co-transfected with the reporter containing the 1.2 kb DNA fragment downstream of P2 as compared to the cells transfected with empty vector or wtFANCD2 in various combinations, all of which showed a similar basal level of luciferase activity (Figure 3B; data not shown). These results suggest that inactivated FANCD2 may play a role in enhancing the transcription of ΔNp63, which appears to be a new function for the inactivated FANCD2, rather than a loss function of wtFANCD2. To support the reporter activity observed, we conducted ChIP analysis on the binding potential of inactivated FANCD2 protein to the 1.2 kb DNA fragment. We found that antibodies against inactivated FANCD2 can also pull down a substantial amount of the downstream DNA fragment, but not the one upstream of the P2 promoter (Figure 3C), which agrees with the reporter assay (Figure 3B). Therefore, inactivated FANCD2 can regulate ΔNp63 mRNA expression through the association with a DNA sequence downstream of the known P2 promoter of ΔNp63.

Bottom Line: Here we unexpectedly found that ΔNp63 mRNA was expressed at high levels in human cancer cells carrying an impaired FA pathway compared to the corresponding control cells carrying an intact FA pathway.In vivo, the elevation of FAVL, a tumor promotion factor that inhibits FANCD2 activation, was found to be positively associated with ΔNp63 expression in human cancer tissues.Collectively, these results document a novel role of an inactivated FANCD2 in upregulating ΔNp63, advancing our understanding of how an impaired FA pathway contributes to the pathogenesis of human cancer.

View Article: PubMed Central - PubMed

Affiliation: University of Hawaii Cancer Center, University of Hawaii, Honolulu, HI, USA.

ABSTRACT
A compromised Fanconi Anemia (FA) signaling pathway, often resulting from an inactivated FANCD2, was recently recognized to contribute to the development of non-FA human tumors. However, it is largely unknown as to how an impaired FA pathway or an inactivated FANCD2 promotes tumorigenesis. Here we unexpectedly found that ΔNp63 mRNA was expressed at high levels in human cancer cells carrying an impaired FA pathway compared to the corresponding control cells carrying an intact FA pathway. This observation was recapitulated upon conditionally managing the status of FANCD2 monoubiquitination /activation in 293T cells. Importantly, ΔNp63 elevation upon FANCD2 inactivation was confirmed in human fibroblasts derived from FA patients. Moreover, we have identified a 189 bp DNA fragment downstream of the ΔNp63 promoter (P2) that can mediate the upregulation of ΔNp63 by an inactivated FANCD2, and determined that elevated ΔNp63 is high enough to promote cancer cell proliferation and metastasis. In vivo, the elevation of FAVL, a tumor promotion factor that inhibits FANCD2 activation, was found to be positively associated with ΔNp63 expression in human cancer tissues. Collectively, these results document a novel role of an inactivated FANCD2 in upregulating ΔNp63, advancing our understanding of how an impaired FA pathway contributes to the pathogenesis of human cancer.

Show MeSH
Related in: MedlinePlus