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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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ΔNp63 elevation is recapitulated in 293T cells and FA patient cells upon the inactivated FANCD2(A) A higher level of ΔNp63 expression follows FAVL elevation in a Tet-on inducible expression system. The 293T conditional expression cell line was established to express FAVL controlled by the inducer, Doxycycline (Dox). 0.5 or 1 μg/ml Dox addition can induce FAVL expression at a higher level than non-treated cells (0 μg/ml Dox). Under the same conditions, the levels of ΔNp63 expression are elevated and the basal levels of FANCD2 monoubiquitination are reduced (monoubiquitinated/non-ubiquitinated ratio is decreased). The conditional overexpression of FAVL that provides an impaired FA pathway was correlated with the corresponding elevation of ΔNp63 expression (NIH Image J software was used to evaluate the band densities, with which the relative FAVL and ΔNp63 expression levels were calculated upon the corresponding control =1 for non-induced cells. monoubiquitinated/non-ubiquitinated ratio was generated also upon the band densities). (B) ΔNp63 mRNA expression is only detectable in PD220 cells (FANCA−/−), but not in PD20 (FANCD2−/−) and PD20+FANCD2 cells. Total RNA was isolated from normally growing PD220, PD20 and PD20+FANCD2 cell. The expression of ΔNp63 mRNA was detected by RT-PCR, which was clearly detectable in PD220 (FANCA−/−), and not in PD20 (FANCD2−/−) or PD20+FANCD2 cells. The un-monoubiquitinated FANCD2, but not the loss of FANCD2, is able to elevate ΔNp63 expression. [Two of three separated RT-PCR results are shown, without (top panel) or with (bottom panel) control actin amplification in the same PCR reaction].
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Figure 2: ΔNp63 elevation is recapitulated in 293T cells and FA patient cells upon the inactivated FANCD2(A) A higher level of ΔNp63 expression follows FAVL elevation in a Tet-on inducible expression system. The 293T conditional expression cell line was established to express FAVL controlled by the inducer, Doxycycline (Dox). 0.5 or 1 μg/ml Dox addition can induce FAVL expression at a higher level than non-treated cells (0 μg/ml Dox). Under the same conditions, the levels of ΔNp63 expression are elevated and the basal levels of FANCD2 monoubiquitination are reduced (monoubiquitinated/non-ubiquitinated ratio is decreased). The conditional overexpression of FAVL that provides an impaired FA pathway was correlated with the corresponding elevation of ΔNp63 expression (NIH Image J software was used to evaluate the band densities, with which the relative FAVL and ΔNp63 expression levels were calculated upon the corresponding control =1 for non-induced cells. monoubiquitinated/non-ubiquitinated ratio was generated also upon the band densities). (B) ΔNp63 mRNA expression is only detectable in PD220 cells (FANCA−/−), but not in PD20 (FANCD2−/−) and PD20+FANCD2 cells. Total RNA was isolated from normally growing PD220, PD20 and PD20+FANCD2 cell. The expression of ΔNp63 mRNA was detected by RT-PCR, which was clearly detectable in PD220 (FANCA−/−), and not in PD20 (FANCD2−/−) or PD20+FANCD2 cells. The un-monoubiquitinated FANCD2, but not the loss of FANCD2, is able to elevate ΔNp63 expression. [Two of three separated RT-PCR results are shown, without (top panel) or with (bottom panel) control actin amplification in the same PCR reaction].

Mentions: To validate the relationship between an impaired status of the FA pathway and the expression levels of ΔNp63, we tested the association between the levels of ΔNp63 expression and an impaired FA status in non-tumorigenic 293T cells wherein the genetic background is relatively closer to normal cells as compared to the tested tumor cells above (HCT116, U2OS and HTB-4). We generated Tet-on inducible 293T stably-transfected cells, within which FANCD2 is only inactivated when FAVL is overexpressed, controlled by the conditional inducer, Doxycycline (Dox, a more stable tetracycline analogue). We found that the level of ΔNp63 expression is correspondingly elevated when the level of FAVL expression is increased (Figure 2A). This observation confirms the above finding (Figure 1), indicating that the regulation of ΔNp63 expression by inactivated FANCD2 is not restricted to tumor cells. We further validated the association of ΔNp63 expression with inactivated FANCD2 by using FA patient cells, in which the variables only result from the FANCD2 status. We examined the level of ΔNp63 mRNA expression in PD20 (FANCD2−/−), PD20+FANCD2, and PD220 (FANCA−/−; carrying an unstable E3 ubiquitin ligase complex, leading to an un-monoubiquitinated FANCD2). We found that the ΔNp63 mRNA expression level is only detectable in PD220 cells in which FANCD2 is inactivated, but not in PD20 cells with or without a reconstituted wtFANCD2 (Figure 2B). Collectively, ΔNp63 elevation is associated with an impaired status of the FA pathway, and it may act as a tumorigenic mediator of inactivated FANCD2 during tumor development.


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)

ΔNp63 elevation is recapitulated in 293T cells and FA patient cells upon the inactivated FANCD2(A) A higher level of ΔNp63 expression follows FAVL elevation in a Tet-on inducible expression system. The 293T conditional expression cell line was established to express FAVL controlled by the inducer, Doxycycline (Dox). 0.5 or 1 μg/ml Dox addition can induce FAVL expression at a higher level than non-treated cells (0 μg/ml Dox). Under the same conditions, the levels of ΔNp63 expression are elevated and the basal levels of FANCD2 monoubiquitination are reduced (monoubiquitinated/non-ubiquitinated ratio is decreased). The conditional overexpression of FAVL that provides an impaired FA pathway was correlated with the corresponding elevation of ΔNp63 expression (NIH Image J software was used to evaluate the band densities, with which the relative FAVL and ΔNp63 expression levels were calculated upon the corresponding control =1 for non-induced cells. monoubiquitinated/non-ubiquitinated ratio was generated also upon the band densities). (B) ΔNp63 mRNA expression is only detectable in PD220 cells (FANCA−/−), but not in PD20 (FANCD2−/−) and PD20+FANCD2 cells. Total RNA was isolated from normally growing PD220, PD20 and PD20+FANCD2 cell. The expression of ΔNp63 mRNA was detected by RT-PCR, which was clearly detectable in PD220 (FANCA−/−), and not in PD20 (FANCD2−/−) or PD20+FANCD2 cells. The un-monoubiquitinated FANCD2, but not the loss of FANCD2, is able to elevate ΔNp63 expression. [Two of three separated RT-PCR results are shown, without (top panel) or with (bottom panel) control actin amplification in the same PCR reaction].
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Figure 2: ΔNp63 elevation is recapitulated in 293T cells and FA patient cells upon the inactivated FANCD2(A) A higher level of ΔNp63 expression follows FAVL elevation in a Tet-on inducible expression system. The 293T conditional expression cell line was established to express FAVL controlled by the inducer, Doxycycline (Dox). 0.5 or 1 μg/ml Dox addition can induce FAVL expression at a higher level than non-treated cells (0 μg/ml Dox). Under the same conditions, the levels of ΔNp63 expression are elevated and the basal levels of FANCD2 monoubiquitination are reduced (monoubiquitinated/non-ubiquitinated ratio is decreased). The conditional overexpression of FAVL that provides an impaired FA pathway was correlated with the corresponding elevation of ΔNp63 expression (NIH Image J software was used to evaluate the band densities, with which the relative FAVL and ΔNp63 expression levels were calculated upon the corresponding control =1 for non-induced cells. monoubiquitinated/non-ubiquitinated ratio was generated also upon the band densities). (B) ΔNp63 mRNA expression is only detectable in PD220 cells (FANCA−/−), but not in PD20 (FANCD2−/−) and PD20+FANCD2 cells. Total RNA was isolated from normally growing PD220, PD20 and PD20+FANCD2 cell. The expression of ΔNp63 mRNA was detected by RT-PCR, which was clearly detectable in PD220 (FANCA−/−), and not in PD20 (FANCD2−/−) or PD20+FANCD2 cells. The un-monoubiquitinated FANCD2, but not the loss of FANCD2, is able to elevate ΔNp63 expression. [Two of three separated RT-PCR results are shown, without (top panel) or with (bottom panel) control actin amplification in the same PCR reaction].
Mentions: To validate the relationship between an impaired status of the FA pathway and the expression levels of ΔNp63, we tested the association between the levels of ΔNp63 expression and an impaired FA status in non-tumorigenic 293T cells wherein the genetic background is relatively closer to normal cells as compared to the tested tumor cells above (HCT116, U2OS and HTB-4). We generated Tet-on inducible 293T stably-transfected cells, within which FANCD2 is only inactivated when FAVL is overexpressed, controlled by the conditional inducer, Doxycycline (Dox, a more stable tetracycline analogue). We found that the level of ΔNp63 expression is correspondingly elevated when the level of FAVL expression is increased (Figure 2A). This observation confirms the above finding (Figure 1), indicating that the regulation of ΔNp63 expression by inactivated FANCD2 is not restricted to tumor cells. We further validated the association of ΔNp63 expression with inactivated FANCD2 by using FA patient cells, in which the variables only result from the FANCD2 status. We examined the level of ΔNp63 mRNA expression in PD20 (FANCD2−/−), PD20+FANCD2, and PD220 (FANCA−/−; carrying an unstable E3 ubiquitin ligase complex, leading to an un-monoubiquitinated FANCD2). We found that the ΔNp63 mRNA expression level is only detectable in PD220 cells in which FANCD2 is inactivated, but not in PD20 cells with or without a reconstituted wtFANCD2 (Figure 2B). Collectively, ΔNp63 elevation is associated with an impaired status of the FA pathway, and it may act as a tumorigenic mediator of inactivated FANCD2 during tumor development.

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