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

ΔNp63 expression is elevated in cancer cells carrying an inactivated FA pathway/ inactivated FANCD2 under normoxic and hypoxic conditionsSets of HCT116, U2OS, and HTB-4 stably-transfected cell pairs isogenic to the status of the FA pathway were used to detect ΔNp63 mRNA and protein levels. Levels of both ΔNp63 mRNA and protein are higher in cells carrying an impaired FA pathway compared to the corresponding empty vector-containing control cells (carrying an intact FA pathway) under normal growth conditions or the treatment with 280 μM of Deferoxamine (DFO, a hypoxia-mimicking drug). (A) HCT116 colon cancer and (B) Human osteosarcoma U2OS cells stably express a down-regulated FANCL (Supplementary Figure 1). (C) HTB-4 bladder cancer cells stably express a higher level of FAVL and carry an impaired FA pathway [18]. (NIH Image J software was used to evaluate the band densities, with which the ΔNp63 expression levels were calculated upon the corresponding control =1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: ΔNp63 expression is elevated in cancer cells carrying an inactivated FA pathway/ inactivated FANCD2 under normoxic and hypoxic conditionsSets of HCT116, U2OS, and HTB-4 stably-transfected cell pairs isogenic to the status of the FA pathway were used to detect ΔNp63 mRNA and protein levels. Levels of both ΔNp63 mRNA and protein are higher in cells carrying an impaired FA pathway compared to the corresponding empty vector-containing control cells (carrying an intact FA pathway) under normal growth conditions or the treatment with 280 μM of Deferoxamine (DFO, a hypoxia-mimicking drug). (A) HCT116 colon cancer and (B) Human osteosarcoma U2OS cells stably express a down-regulated FANCL (Supplementary Figure 1). (C) HTB-4 bladder cancer cells stably express a higher level of FAVL and carry an impaired FA pathway [18]. (NIH Image J software was used to evaluate the band densities, with which the ΔNp63 expression levels were calculated upon the corresponding control =1).

Mentions: ΔNp63 is often highly expressed in a variety of human cancers, including squamous cell origin (head and neck, lung), breast and bladder cancers [8]. Coincidently, there is a high incidence of a variety of human cancers associated with FA patients, especially head and neck cancers [22]. We therefore asked whether an impaired FA signaling pathway has any relation to the high expression levels of ΔNp63. We generated two sets of stably-transfected cell pairs (Supplementary Figure 1), isogenic to the level of FANCL expression that would result in an impaired or intact status of the FA pathway [23], to examine how the status of the FA pathway is relevant to the level of ΔNp63 expression. Surprisingly, under both normal and hypoxic growth conditions (the latter is induced by a hypoxic mimicking drug), ΔNp63 mRNA and protein expression levels were found to be elevated in U2OS and HCT116 cells carrying an impaired FA pathway as compared to the corresponding empty vector-transfected control cells, in which the FA pathway is intact (Figures 1A and B). To confirm that ΔNp63 elevation results from an inactivated FANCD2, not from the off-target effect of FANCL silencing, we detected ΔNp63 expression in stably-transfected cell pairs derived from the HTB-4 bladder cancer cell line, in which the impaired FA pathway was induced by a high expression level of FAVL (a variant of FANCL that acts as a tumor promotion factor by inactivating FANCD2) [17, 18]. We found that HTB-4 cells carrying an inactivated FANCD2 do express ΔNp63 at a higher level in comparison with the corresponding control HTB-4 cells (Figure 1C). Together, ΔNp63 appears to be a downstream target of inactivated FANCD2 not only under the normal cell growth condition but also under conditions of hypoxia. This suggests that the regulation of ΔNp63 expression by inactivated FANCD2 may play an important role in the development of human tumors, presumably starting from tumor initiation to tumor mass development, which often leads to hypoxic conditions within solid tumors.


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 expression is elevated in cancer cells carrying an inactivated FA pathway/ inactivated FANCD2 under normoxic and hypoxic conditionsSets of HCT116, U2OS, and HTB-4 stably-transfected cell pairs isogenic to the status of the FA pathway were used to detect ΔNp63 mRNA and protein levels. Levels of both ΔNp63 mRNA and protein are higher in cells carrying an impaired FA pathway compared to the corresponding empty vector-containing control cells (carrying an intact FA pathway) under normal growth conditions or the treatment with 280 μM of Deferoxamine (DFO, a hypoxia-mimicking drug). (A) HCT116 colon cancer and (B) Human osteosarcoma U2OS cells stably express a down-regulated FANCL (Supplementary Figure 1). (C) HTB-4 bladder cancer cells stably express a higher level of FAVL and carry an impaired FA pathway [18]. (NIH Image J software was used to evaluate the band densities, with which the ΔNp63 expression levels were calculated upon the corresponding control =1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: ΔNp63 expression is elevated in cancer cells carrying an inactivated FA pathway/ inactivated FANCD2 under normoxic and hypoxic conditionsSets of HCT116, U2OS, and HTB-4 stably-transfected cell pairs isogenic to the status of the FA pathway were used to detect ΔNp63 mRNA and protein levels. Levels of both ΔNp63 mRNA and protein are higher in cells carrying an impaired FA pathway compared to the corresponding empty vector-containing control cells (carrying an intact FA pathway) under normal growth conditions or the treatment with 280 μM of Deferoxamine (DFO, a hypoxia-mimicking drug). (A) HCT116 colon cancer and (B) Human osteosarcoma U2OS cells stably express a down-regulated FANCL (Supplementary Figure 1). (C) HTB-4 bladder cancer cells stably express a higher level of FAVL and carry an impaired FA pathway [18]. (NIH Image J software was used to evaluate the band densities, with which the ΔNp63 expression levels were calculated upon the corresponding control =1).
Mentions: ΔNp63 is often highly expressed in a variety of human cancers, including squamous cell origin (head and neck, lung), breast and bladder cancers [8]. Coincidently, there is a high incidence of a variety of human cancers associated with FA patients, especially head and neck cancers [22]. We therefore asked whether an impaired FA signaling pathway has any relation to the high expression levels of ΔNp63. We generated two sets of stably-transfected cell pairs (Supplementary Figure 1), isogenic to the level of FANCL expression that would result in an impaired or intact status of the FA pathway [23], to examine how the status of the FA pathway is relevant to the level of ΔNp63 expression. Surprisingly, under both normal and hypoxic growth conditions (the latter is induced by a hypoxic mimicking drug), ΔNp63 mRNA and protein expression levels were found to be elevated in U2OS and HCT116 cells carrying an impaired FA pathway as compared to the corresponding empty vector-transfected control cells, in which the FA pathway is intact (Figures 1A and B). To confirm that ΔNp63 elevation results from an inactivated FANCD2, not from the off-target effect of FANCL silencing, we detected ΔNp63 expression in stably-transfected cell pairs derived from the HTB-4 bladder cancer cell line, in which the impaired FA pathway was induced by a high expression level of FAVL (a variant of FANCL that acts as a tumor promotion factor by inactivating FANCD2) [17, 18]. We found that HTB-4 cells carrying an inactivated FANCD2 do express ΔNp63 at a higher level in comparison with the corresponding control HTB-4 cells (Figure 1C). Together, ΔNp63 appears to be a downstream target of inactivated FANCD2 not only under the normal cell growth condition but also under conditions of hypoxia. This suggests that the regulation of ΔNp63 expression by inactivated FANCD2 may play an important role in the development of human tumors, presumably starting from tumor initiation to tumor mass development, which often leads to hypoxic conditions within solid tumors.

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