Limits...
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.

Show MeSH

Related in: MedlinePlus

A 441bp DNA sequence within the 1.2kb fragemtn can mediate the regulation of ΔNp63 expression by inactivated FANCD2(A) The 1.2 kb DNA fragment was divided into three DNA segments with a size of 377, 401, or 441 bps. (B) 441 bp DNA fragment, but not the 377 and 403 bp ones (Supplementary Figure 2, left panel), can be pulled down more by FANCD2 antibodies in U2OS and HCT116 stably-transfected cells carrying an inactivated FANCD2 compared to corresponding control cells with an intact FA pathway. (C) the 441 bp DNA fragment can also be pulled down more by Flag antibodies in cells transiently co-transfected with Flag-mtFANCD2, but not Flag-wtFANCD2. A ChIP assay was performed in cells transiently transfected with pEGFP-Flag wtFANCD2 or mtFANCD2 by using Flag antibodies. ChIP-PCR primers were designed for three fragments shown in (A). The similar transfection efficiency was measured through western blotting by using antibodies against GFP or Flag (right panel). (D) K561R mtFANCD2 stimulates the 441-reporter activity. Similarly as previously done, three DNA segments were individually cloned into the upstream of pGL-3-promoter-reporter, and named the 377, 403, or 441 -reporters. 293T cells were transiently transfected respectively with these reporters and pEGFP-Flag plasmids containing wt or mtFNACD2. All luciferase assays were normalized for transfection efficiency by renilla reporter activity. Western blot analysis with Flag or GFP antibodies was performed to verify expressions of both wt and mtFANCD2 proteins. The results shown are a representative of five independent experiments performed each time in triplicate, error bars indicating the standard deviation [the 377-reporter (not shown) produces luciferase activity similar to the one derived from the 403-reporter].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3824532&req=5

Figure 4: A 441bp DNA sequence within the 1.2kb fragemtn can mediate the regulation of ΔNp63 expression by inactivated FANCD2(A) The 1.2 kb DNA fragment was divided into three DNA segments with a size of 377, 401, or 441 bps. (B) 441 bp DNA fragment, but not the 377 and 403 bp ones (Supplementary Figure 2, left panel), can be pulled down more by FANCD2 antibodies in U2OS and HCT116 stably-transfected cells carrying an inactivated FANCD2 compared to corresponding control cells with an intact FA pathway. (C) the 441 bp DNA fragment can also be pulled down more by Flag antibodies in cells transiently co-transfected with Flag-mtFANCD2, but not Flag-wtFANCD2. A ChIP assay was performed in cells transiently transfected with pEGFP-Flag wtFANCD2 or mtFANCD2 by using Flag antibodies. ChIP-PCR primers were designed for three fragments shown in (A). The similar transfection efficiency was measured through western blotting by using antibodies against GFP or Flag (right panel). (D) K561R mtFANCD2 stimulates the 441-reporter activity. Similarly as previously done, three DNA segments were individually cloned into the upstream of pGL-3-promoter-reporter, and named the 377, 403, or 441 -reporters. 293T cells were transiently transfected respectively with these reporters and pEGFP-Flag plasmids containing wt or mtFNACD2. All luciferase assays were normalized for transfection efficiency by renilla reporter activity. Western blot analysis with Flag or GFP antibodies was performed to verify expressions of both wt and mtFANCD2 proteins. The results shown are a representative of five independent experiments performed each time in triplicate, error bars indicating the standard deviation [the 377-reporter (not shown) produces luciferase activity similar to the one derived from the 403-reporter].

Mentions: Next, we wanted to narrow down the specific region within the 1.2 kb DNA fragment that mediates the transactivation activity of the inactivated FANCD2 (Figures 1-3). By dividing the 1.2 kb DNA fragment into three segments with a size of about 400 bp each (Figure 4A), we designed three sets of primers and performed ChIP assays by using U2OS and HCT116 stably-transfected cell pairs carrying activatible or in-activatible FANCD2. We found that the third 441 bp DNA fragment (Figure 4A) can be pulled down more along with the FANCD2 protein from the lysates prepared from cells carrying in-activatible FANCD2 (Figure 4B; Supplementary Figure 2). This was further validated in 293T cells transiently transfected with Flag-wt or mtFANCD2 plasmids. As shown in Figure 3C, a greater pull-down of the 441 bp fragment by Flag antibodies was seen in the lysates prepared from 293T cells containing Flag-mtFANCD2 compared to the same cells transfected with other reporters. Correspondingly, we generated three reporter constructs to have an approximately 400 bp DNA fragment located upstream of the SV40 promoter of the pGL-3 luciferase reporter. We found the third fragment-containing reporter (441-reporter) had a higher luciferase activity in cells transfected with mtFANCD2 compared to cells transfected either with wtFANCD2 or the empty vector control. These reporters appeared to have a similar level of luciferase activity, suggesting again that the enhanced reporter activity is initiated by mtFANCD2, but not the loss function of wtFANCD2 (Figure 4D and data not shown). Similarly, we further divided the 441bp DNA fragment (Figure 5A) and found a DNA fragment with a size of 189 bps (Figures 5B-D; Supplementary Figure 2), which can mediate the enhanced reporter activity initiated by inactivated FANCD2 as well as the in vivo association with inactivated FANCD2. Taken together, the 189 bp DNA fragment downstream of the P2 promoter is a cis-element that can mediate the upregulation of ΔNp63 expression by inactivated FANCD2 at the genetic level.


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)

A 441bp DNA sequence within the 1.2kb fragemtn can mediate the regulation of ΔNp63 expression by inactivated FANCD2(A) The 1.2 kb DNA fragment was divided into three DNA segments with a size of 377, 401, or 441 bps. (B) 441 bp DNA fragment, but not the 377 and 403 bp ones (Supplementary Figure 2, left panel), can be pulled down more by FANCD2 antibodies in U2OS and HCT116 stably-transfected cells carrying an inactivated FANCD2 compared to corresponding control cells with an intact FA pathway. (C) the 441 bp DNA fragment can also be pulled down more by Flag antibodies in cells transiently co-transfected with Flag-mtFANCD2, but not Flag-wtFANCD2. A ChIP assay was performed in cells transiently transfected with pEGFP-Flag wtFANCD2 or mtFANCD2 by using Flag antibodies. ChIP-PCR primers were designed for three fragments shown in (A). The similar transfection efficiency was measured through western blotting by using antibodies against GFP or Flag (right panel). (D) K561R mtFANCD2 stimulates the 441-reporter activity. Similarly as previously done, three DNA segments were individually cloned into the upstream of pGL-3-promoter-reporter, and named the 377, 403, or 441 -reporters. 293T cells were transiently transfected respectively with these reporters and pEGFP-Flag plasmids containing wt or mtFNACD2. All luciferase assays were normalized for transfection efficiency by renilla reporter activity. Western blot analysis with Flag or GFP antibodies was performed to verify expressions of both wt and mtFANCD2 proteins. The results shown are a representative of five independent experiments performed each time in triplicate, error bars indicating the standard deviation [the 377-reporter (not shown) produces luciferase activity similar to the one derived from the 403-reporter].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824532&req=5

Figure 4: A 441bp DNA sequence within the 1.2kb fragemtn can mediate the regulation of ΔNp63 expression by inactivated FANCD2(A) The 1.2 kb DNA fragment was divided into three DNA segments with a size of 377, 401, or 441 bps. (B) 441 bp DNA fragment, but not the 377 and 403 bp ones (Supplementary Figure 2, left panel), can be pulled down more by FANCD2 antibodies in U2OS and HCT116 stably-transfected cells carrying an inactivated FANCD2 compared to corresponding control cells with an intact FA pathway. (C) the 441 bp DNA fragment can also be pulled down more by Flag antibodies in cells transiently co-transfected with Flag-mtFANCD2, but not Flag-wtFANCD2. A ChIP assay was performed in cells transiently transfected with pEGFP-Flag wtFANCD2 or mtFANCD2 by using Flag antibodies. ChIP-PCR primers were designed for three fragments shown in (A). The similar transfection efficiency was measured through western blotting by using antibodies against GFP or Flag (right panel). (D) K561R mtFANCD2 stimulates the 441-reporter activity. Similarly as previously done, three DNA segments were individually cloned into the upstream of pGL-3-promoter-reporter, and named the 377, 403, or 441 -reporters. 293T cells were transiently transfected respectively with these reporters and pEGFP-Flag plasmids containing wt or mtFNACD2. All luciferase assays were normalized for transfection efficiency by renilla reporter activity. Western blot analysis with Flag or GFP antibodies was performed to verify expressions of both wt and mtFANCD2 proteins. The results shown are a representative of five independent experiments performed each time in triplicate, error bars indicating the standard deviation [the 377-reporter (not shown) produces luciferase activity similar to the one derived from the 403-reporter].
Mentions: Next, we wanted to narrow down the specific region within the 1.2 kb DNA fragment that mediates the transactivation activity of the inactivated FANCD2 (Figures 1-3). By dividing the 1.2 kb DNA fragment into three segments with a size of about 400 bp each (Figure 4A), we designed three sets of primers and performed ChIP assays by using U2OS and HCT116 stably-transfected cell pairs carrying activatible or in-activatible FANCD2. We found that the third 441 bp DNA fragment (Figure 4A) can be pulled down more along with the FANCD2 protein from the lysates prepared from cells carrying in-activatible FANCD2 (Figure 4B; Supplementary Figure 2). This was further validated in 293T cells transiently transfected with Flag-wt or mtFANCD2 plasmids. As shown in Figure 3C, a greater pull-down of the 441 bp fragment by Flag antibodies was seen in the lysates prepared from 293T cells containing Flag-mtFANCD2 compared to the same cells transfected with other reporters. Correspondingly, we generated three reporter constructs to have an approximately 400 bp DNA fragment located upstream of the SV40 promoter of the pGL-3 luciferase reporter. We found the third fragment-containing reporter (441-reporter) had a higher luciferase activity in cells transfected with mtFANCD2 compared to cells transfected either with wtFANCD2 or the empty vector control. These reporters appeared to have a similar level of luciferase activity, suggesting again that the enhanced reporter activity is initiated by mtFANCD2, but not the loss function of wtFANCD2 (Figure 4D and data not shown). Similarly, we further divided the 441bp DNA fragment (Figure 5A) and found a DNA fragment with a size of 189 bps (Figures 5B-D; Supplementary Figure 2), which can mediate the enhanced reporter activity initiated by inactivated FANCD2 as well as the in vivo association with inactivated FANCD2. Taken together, the 189 bp DNA fragment downstream of the P2 promoter is a cis-element that can mediate the upregulation of ΔNp63 expression by inactivated FANCD2 at the genetic level.

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