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.     

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.     

ΔNp63α Levels Correlate with Clinical Tumor Response to Cisplatin

After exposure to damaging agents, the p53 tumor suppressor is stabilized mediating cell cycle arrest and apoptosis. p53 family member, ΔNp63α promotes cell proliferation and accelerates tumor growth. We previously found that the genotoxic stress agents induced a decrease of ΔNp63α . We further observed that genotoxic stress mediated phosphorylation of ΔNp63α targeting it into proteasome degradation. Here, we found that high ΔNp63 protein levels in primary tumors accurately predicted response to platinum based chemotherapy and a favorable outcome in head and neck cancer patients. Our data suggest that degradation of ΔNp63α is part of the cellular response to DNA damage in head and neck cancers. The findings may have implications for the rational use of DNA damaging agents in human cancer.     

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.     

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.     

p38α: A Suppressor of Cell Proliferation and Tumorigenesis

The mitogen-activated protein kinase (MAPK) p38! is involved in numerous biological processes and is a drug target for inflammation-associated diseases. Genetic analysis in mice demonstrated that fetuses lacking p38! are embryonic lethal owing to impaired placental development. The function of p38! in mice after birth remained unclear until conditional alleles of p38α were used. It was found that p38α is essential for lung functionin both neonatal and adult mice. Increased proliferation and impaired differentiation are the hallmarks of p38α-deficient cells. Moreover, mice deficient in p38α are prone to cancerdevelopment using carcinogen or oncogene-induced cancer models. p38α can suppress cell proliferation by antagonizing the JNK/c-Jun pathway, which is an important regulator of proliferation and apoptosis. These findings suggest that therapeutic inhibition of p38 might lead to unwanted proliferation. Therefore, a combined inhibition of p38 and other pathways, such as the JNK pathway, should be considered for targeting cancer inflammation.     

A Novel Role of p38α MAPK in Mitotic Progression Independent of Its Kinase Activity

Activation of p38α MAPK triggers G2/M checkpoint, thus inhibiting cell proliferation. In this study we found that depletion of p38α by RNAi also inhibited cell proliferation and caused mitotic arrest. However, treatment with selective small molecule p38 kinase inhibitors had no effect on cell cycle progression, even though the p38 kinase was completely inhibited, revealing p38α functions that are independent of its kinase activity. Indeed, ectopic expression of a kinase negative p38α rescued the lethality caused by RNAi-depletion of the endogenous p38α, thus providing further evidence for a kinase-independent function of p38α. In addition, we showed that overexpression of the wild type or kinase-negative p38α also strongly inhibited cell proliferation, similarly as RNAi depletion of p38α. Together the results demonstrate that, in addition to its kinase-dependent functions, such as in activation of G2/M checkpoint, p38α also has an essential, kinase-independent function.     

Regulation of the Cyclin-Dependent Kinase Inhibitor p57Kip2 Expression by p63

The cyclin-dependent kinase (CDK) inhibitor p57^Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57^Kip2 gene was recognized as a target gene for p73β, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knock out mice do not resemble each other while there is a phenotypic overlap betweeen the p57^Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57^Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the δNp63α protein is associated to three different regions of the p57Kip2 gene. δNp63 can activate both the endogenous p57^Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57^Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57^Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57^Kip2 expression and that this regulation is subverted in AEC p63 mutants.