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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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Silencing ΔNp63 mRNA expression substantially abrogates the tumorigenic potential of HTB-4 bladder cancer cells triggered by FA pathway impaired by elevated FAVL(A) Silencing ΔNp63 blocks the advantage in cell proliferation triggered by FAVL elevation under both hypoxia and normoxia (normal oxygen). HTB-4+EV or +FAVL stably-transfected cells with or without silenced ΔNp63 (Supplementary Figure 3) were plated at day 0 with an equal number. The total cell number was recounted on day 5 and plotted in the left panel for the relative cell growth. The representative images on day 5 were shown for cells growing under both normoxia and hypoxic stress (right panel). (B) Silencing ΔNp63 blocks the invasive advantage triggered by FAVL elevation. As performed previously [11], two sets of stably-transfected cell pairs (HTB-4+EV+Lenti-EV & HTB-4+EV+Lenti-shRNAi; HTB-4+FAVL+Lenti+EV&HTB-4+FAVL+Lenti-shRNAi) (Supplementary Figure 3) were grown in 140 μm DFO overnight and plated into transwells the following day in medium containing 140 μm DFO. Transwells were processed accordingly 2 hours after plating. Cells were counted with 10 random fields at a magnification of 400X. All counted cell numbers were used to plot the relative invasive potential (right panel). Representative images of the invasive cell density are shown in the left panel. Cells expressing elevated FAVL have a stronger invasive potential (image panel-3 compared to image panel-1). Silencing ΔNp63 can mitigate the invasive potential of cells carrying FAVL (image panel 4, compared to image panel 3), but not cells without FAVL elevation (image panel 2 compared to image panel-1; SD was made from three separate experiments).
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Figure 6: Silencing ΔNp63 mRNA expression substantially abrogates the tumorigenic potential of HTB-4 bladder cancer cells triggered by FA pathway impaired by elevated FAVL(A) Silencing ΔNp63 blocks the advantage in cell proliferation triggered by FAVL elevation under both hypoxia and normoxia (normal oxygen). HTB-4+EV or +FAVL stably-transfected cells with or without silenced ΔNp63 (Supplementary Figure 3) were plated at day 0 with an equal number. The total cell number was recounted on day 5 and plotted in the left panel for the relative cell growth. The representative images on day 5 were shown for cells growing under both normoxia and hypoxic stress (right panel). (B) Silencing ΔNp63 blocks the invasive advantage triggered by FAVL elevation. As performed previously [11], two sets of stably-transfected cell pairs (HTB-4+EV+Lenti-EV & HTB-4+EV+Lenti-shRNAi; HTB-4+FAVL+Lenti+EV&HTB-4+FAVL+Lenti-shRNAi) (Supplementary Figure 3) were grown in 140 μm DFO overnight and plated into transwells the following day in medium containing 140 μm DFO. Transwells were processed accordingly 2 hours after plating. Cells were counted with 10 random fields at a magnification of 400X. All counted cell numbers were used to plot the relative invasive potential (right panel). Representative images of the invasive cell density are shown in the left panel. Cells expressing elevated FAVL have a stronger invasive potential (image panel-3 compared to image panel-1). Silencing ΔNp63 can mitigate the invasive potential of cells carrying FAVL (image panel 4, compared to image panel 3), but not cells without FAVL elevation (image panel 2 compared to image panel-1; SD was made from three separate experiments).

Mentions: Our study thus far reveals that the regulation of ΔNp63 expression by an inactivated FANCD2 may play important roles in the tumor promotion potential resulting from an impaired FA pathway. Whether the gained growth potential of cells harboring an impaired FA pathway is partly attributed to ΔNp63 needs to be tested. We examined the growth potential of FAVL-elevated HTB-4 cells with or without silencing ΔNp63 expression (Supplementary Figure 3). As shown in Figure 6A, the gained growth potential resulting from the elevated FAVL can be mitigated by silencing ΔNp63 expression, but there is no noticeable difference between empty vector-containing HTB-4 cells with or without silenced ΔNp63. Furthermore, the metastatic potential resulting from FAVL elevation also shows a similar pattern of change (Figure 6B). These results demonstrate that inactivated FANCD2 is not only able to upregulate ΔNp63 expression, but also that elevated ΔNp63 expression can contribute to tumor development through, at least, enhancing the potentials of cell proliferation and metastasis. We next explored the clinical importance of upregulation of ΔNp63 by inactivated FANCD2. Using immunohistochemistry, we found the level of FAVL expression (cytoplasmic staining) is positively associated with the level of ΔNp63 protein expression (nuclear staining) in 25 human bladder tissue samples tested (Figure 7A; Supplementary table 1). Taken together, both in vitro and in vivo data reveal that ΔNp63 is a functional mediator of an impaired FA pathway triggered by FANCD2 inactivation, but not by the loss of activated FANCD2. This indicates that the tumorigenicity of inactivated FANCD2 in human tumors, such as human bladder cancer, is at least partly attributed to the subsequent elevation of ΔNp63 (Figure 7B).


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)

Silencing ΔNp63 mRNA expression substantially abrogates the tumorigenic potential of HTB-4 bladder cancer cells triggered by FA pathway impaired by elevated FAVL(A) Silencing ΔNp63 blocks the advantage in cell proliferation triggered by FAVL elevation under both hypoxia and normoxia (normal oxygen). HTB-4+EV or +FAVL stably-transfected cells with or without silenced ΔNp63 (Supplementary Figure 3) were plated at day 0 with an equal number. The total cell number was recounted on day 5 and plotted in the left panel for the relative cell growth. The representative images on day 5 were shown for cells growing under both normoxia and hypoxic stress (right panel). (B) Silencing ΔNp63 blocks the invasive advantage triggered by FAVL elevation. As performed previously [11], two sets of stably-transfected cell pairs (HTB-4+EV+Lenti-EV & HTB-4+EV+Lenti-shRNAi; HTB-4+FAVL+Lenti+EV&HTB-4+FAVL+Lenti-shRNAi) (Supplementary Figure 3) were grown in 140 μm DFO overnight and plated into transwells the following day in medium containing 140 μm DFO. Transwells were processed accordingly 2 hours after plating. Cells were counted with 10 random fields at a magnification of 400X. All counted cell numbers were used to plot the relative invasive potential (right panel). Representative images of the invasive cell density are shown in the left panel. Cells expressing elevated FAVL have a stronger invasive potential (image panel-3 compared to image panel-1). Silencing ΔNp63 can mitigate the invasive potential of cells carrying FAVL (image panel 4, compared to image panel 3), but not cells without FAVL elevation (image panel 2 compared to image panel-1; SD was made from three separate experiments).
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Figure 6: Silencing ΔNp63 mRNA expression substantially abrogates the tumorigenic potential of HTB-4 bladder cancer cells triggered by FA pathway impaired by elevated FAVL(A) Silencing ΔNp63 blocks the advantage in cell proliferation triggered by FAVL elevation under both hypoxia and normoxia (normal oxygen). HTB-4+EV or +FAVL stably-transfected cells with or without silenced ΔNp63 (Supplementary Figure 3) were plated at day 0 with an equal number. The total cell number was recounted on day 5 and plotted in the left panel for the relative cell growth. The representative images on day 5 were shown for cells growing under both normoxia and hypoxic stress (right panel). (B) Silencing ΔNp63 blocks the invasive advantage triggered by FAVL elevation. As performed previously [11], two sets of stably-transfected cell pairs (HTB-4+EV+Lenti-EV & HTB-4+EV+Lenti-shRNAi; HTB-4+FAVL+Lenti+EV&HTB-4+FAVL+Lenti-shRNAi) (Supplementary Figure 3) were grown in 140 μm DFO overnight and plated into transwells the following day in medium containing 140 μm DFO. Transwells were processed accordingly 2 hours after plating. Cells were counted with 10 random fields at a magnification of 400X. All counted cell numbers were used to plot the relative invasive potential (right panel). Representative images of the invasive cell density are shown in the left panel. Cells expressing elevated FAVL have a stronger invasive potential (image panel-3 compared to image panel-1). Silencing ΔNp63 can mitigate the invasive potential of cells carrying FAVL (image panel 4, compared to image panel 3), but not cells without FAVL elevation (image panel 2 compared to image panel-1; SD was made from three separate experiments).
Mentions: Our study thus far reveals that the regulation of ΔNp63 expression by an inactivated FANCD2 may play important roles in the tumor promotion potential resulting from an impaired FA pathway. Whether the gained growth potential of cells harboring an impaired FA pathway is partly attributed to ΔNp63 needs to be tested. We examined the growth potential of FAVL-elevated HTB-4 cells with or without silencing ΔNp63 expression (Supplementary Figure 3). As shown in Figure 6A, the gained growth potential resulting from the elevated FAVL can be mitigated by silencing ΔNp63 expression, but there is no noticeable difference between empty vector-containing HTB-4 cells with or without silenced ΔNp63. Furthermore, the metastatic potential resulting from FAVL elevation also shows a similar pattern of change (Figure 6B). These results demonstrate that inactivated FANCD2 is not only able to upregulate ΔNp63 expression, but also that elevated ΔNp63 expression can contribute to tumor development through, at least, enhancing the potentials of cell proliferation and metastasis. We next explored the clinical importance of upregulation of ΔNp63 by inactivated FANCD2. Using immunohistochemistry, we found the level of FAVL expression (cytoplasmic staining) is positively associated with the level of ΔNp63 protein expression (nuclear staining) in 25 human bladder tissue samples tested (Figure 7A; Supplementary table 1). Taken together, both in vitro and in vivo data reveal that ΔNp63 is a functional mediator of an impaired FA pathway triggered by FANCD2 inactivation, but not by the loss of activated FANCD2. This indicates that the tumorigenicity of inactivated FANCD2 in human tumors, such as human bladder cancer, is at least partly attributed to the subsequent elevation of ΔNp63 (Figure 7B).

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