RACK1 and Stratifin Target ΔNp63α for a Proteasome Degradation in Head and Neck Squamous Cell Carcinoma Cells upon DNA Damage

P53 family members with a transactivation domain induce cell cycle arrest and promoteapoptosis. However, ΔNp63 isotypes lacking the transactivation (TA)- domain promote cellproliferation and tumorigenesis in vitro and in vivo. Although p53, TAp63 or TAp73 are stabilizedupon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease andphosphorylation of ΔNp63α in squamous cell carcinoma cells. Further work revealed that RACK1physically associated with the p63α C-terminal domain through its WD40 domain. However,stratifin binds with phosphorylated ΔNp63α in response to cisplatin. Upon DNA damage inducedby cisplatin, stratifin mediated a nuclear export of ΔNp63α into cytoplasm and then RACK1targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase.Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and proteindegradation of ΔNp63α, respectively. Our data suggest that modification and down regulation ofΔNp63α is one of the major determinants of the cellular response to DNA damage in human headand neck cancers.     

Regulation of ΔNp63α by NFκΒ

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NF-κΒ in presence of cisplatin. We find that NF-κΒ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NF-κΒ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NF-κΒ with siRNA-mediated silencing NF-κΒ expression attenuates chemotherapy induced degradation of ΔNp63α . These data demonstrate that NF-κΒ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NF-κΒ may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.     

Regulation of ΔNp63α by NFκB

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be downregulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NFκB in presence of cisplatin. We find that NFκB promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NFκB leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NFκB with siRNA-mediated silencing NFκB expression attenuates chemotherapy induced degradation of ΔNp63α. These data demonstrate that NFκB plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NFκB may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.Key words: ΔNp63α, NFκB, ubiquitination, cisplatin, head and neck cancer     

Differential Enhancement of a Cutaneous HPV Promoter by ΔNP63α, JUN and Mutant p53

The mechanism through which cutaneous papillomaviruses induce lesions is largelyunknown. Ectopic expression of the ΔNP63α isoform highly increased the viral promoteractivity. The co-expression of c-Jun mediated and increased the ΔNP63α activity by bindingto the AP-1 site in an enhancer region of the HPV 20 URR. This strong activation by ΔNP63αis diminished in the presence of wtp53 and abolished by the simultaneous expression of “hotspot”mutant p53 R248W. We demonstrate that c-Jun is responsible for the viral promoteractivation through its direct interaction with both ΔNP63α and wtp53. The down-regulationby p53 mutant R248W is accompanied by reduced protein levels of ΔNP63α andphosphorylated c-Jun. The data presented in this study provide insight into a possiblemechanism through which these cellular proteins may modulate a cutaneous papillomavirusgenome to induce viral replication, latent infection or malignant trasnformation.     

p63 and p73: Life and Death in Squamous Cell Carcinoma

The p53 family member p63 plays an essential role in the developing epithelium, and overexpression of the ΔNp63a isoform is frequently observed in human squamous cell carcinomas (SCCs). These findings have suggested that ΔNp63a might function as an oncogene within squamous epithelial cells. Nevertheless, the mechanism by which ΔNp63a might promote tumorigenesis remains poorly understood, and data from mouse models implies that the p63 locus might in fact function as a tumor suppressor in these same tissues. A recent study using RNA interference in human SCC-derived cell lines shows that ΔNp63a mediates an essential survival function in human SCC cells by virtue of its ability to suppress the pro-apoptotic function of the related p53 family member p73. These findings support an oncogenic role for ΔNp63a and they demonstrate the existence of critical physical and functional interactions between endogenous p53 family members in human cancer. Specific chemotherapeutic agents and future targeted approaches may be able to exploit this pathway to therapeutic advantage.     

ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DeltaNp63alpha associated with its increased phosphorylated status. Since DeltaNp63alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DeltaNp63alpha in HNSCC cells upon cisplatin exposure. We found that DeltaNp63alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397, and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DeltaNp63alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DeltaNp63alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DeltaNp63alpha degradation. Finally, we demonstrated that the DeltaNp63alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DeltaNp63alpha following DNA damage.     

Delta Np63 alpha expression is regulated by the phosphoinositide 3-kinase pathway

p63 is a homologue of p53 that functions to maintain progenitor cell populations in stratified epithelia. Delta Np63 alpha is overexpressed in epithelial cancers and has been shown to have oncogenic properties. We have previously reported that inhibition of epidermal growth factor receptor signaling results in a decrease in Delta Np63 alpha expression. Here, we demonstrate Delta Np63 alpha is a target of the phosphoinositide-3-kinase (PI3K) pathway downstream of the epidermal growth factor receptor. Treatment of keratinocytes with epidermal growth factor results in an increase in Delta Np63 alpha expression at the mRNA level, which is abrogated by inhibition of PI3K but not mitogen-activated protein kinase signaling. Small interfering RNA-mediated knockdown of the p110 beta catalytic subunit of PI3K results in a decrease in Delta Np63 alpha protein levels in keratinocytes. The results presented herein suggest that regulation of Delta Np63 alpha expression by the PI3K pathway plays a critical role in the survival and proliferative capacity of squamous epithelia.     

DNA damage down-regulates ΔNp63α and induces apoptosis independent of wild type p53

The tumor suppressor p53 is pivotal in cell growth arrest and apoptosis upon cellular stresses including DNA damage. Mounting evidence indicates that p63 proteins, which are homologs of p53, are also involved in apoptosis under certain circumstances. In this study, we found that treatment with DNA damage agents leads to down-regulation of ΔNp63α and induces apoptosis in FaDu and HaCat cells carrying mutant p53. Further study shows that DNA damage reduces steady-state mRNA level of ΔNp63α, but has little effect on its protein stability. In addition, knockdown of endogenous ΔNp63α directly induces apoptosis and sensitizes cells to DNA damage, while exogenous expression of ΔNp63α partially confers cellular resistance to DNA damage. Together, these data suggest that DNA damage down-regulates ΔNp63α, which may directly contribute to DNA damage-induced apoptosis.     

A dominant negative form of p63 inhibits apoptosis in a p53-independent manner

Stem cells are a source of differentiated cells in multiple tissues. If genetic alterations occur in stem cells, the problem persists and malignant cancers may arise. DeltaNp63alpha-a homologue of the tumor suppressor p53-is exclusively expressed in proliferating undifferentiated epithelial cells and cancer cells of epidermal origin. Here, we show that DeltaNp63alpha antagonizes DNA damage-induced apoptosis in a p53-independent manner. We found that upon cellular injury, DeltaNp63alpha must be downregulated before apoptotic program can be activated. The 5637 cell line has abundant levels of DeltaNp63alpha and mutant p53, and it is resistant to DNA damage-induced apoptosis. The knockdown of DeltaNp63alpha by RNA interference sensitized these cells to apoptosis upon genotoxic insult. This suggests that DeltaNp63alpha plays an anti-apoptotic role regardless of the p53 status. Considering the frequent mutations of p53 in tumor cells, our results provide important implications for the treatment of cancers in which p63 is amplified.