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.     

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.     

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.     

Loss of ΔNp63α promotes mitotic exit in epithelial cells

Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ΔNp63α is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ΔNp63α in cell cycle regulation. ΔNp63α accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ΔNp63α knockdown promoted mitotic exit. ΔNp63α shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ΔNp63α. Our results suggest that ΔNp63α level is cell cycle-regulated and may play a role in the regulation of mitotic exit.     

ROCK‐dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type‐specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation‐inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine–glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.     

Cell density-dependent acetylation of ΔNp63α is associated with p53-dependent cell cycle arrest

ΔNp63α is a p63 isoform that is predominantly expressed in the epidermal stem cells and in cancer. To find the regulatory pathways of ΔNp63α, we assessed whether ΔNp63α is acetylated and determined the functional implications of acetylation. First, the hinge region of p63 was shown to be acetylated by PCAF, similarly to other p53 family members. Second, acetylation synergistically induced cytoplasmic localization of ΔNp63α. Finally, acetyl-ΔNp63α was induced during high-density culture, suggesting that acetylation of ΔNp63α may reinforce cell cycle arrest upon cell contact. Altogether, these findings suggest that acetylation of ΔNp63α contributes to the epidermal homeostasis.     

ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis

The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in?vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process.     

ΔNp63α regulates keratinocyte proliferation by controlling PTEN expression and localization

ΔNp63α, implicated as an oncogene, is upregulated by activated Akt, part of a well-known cell survival pathway. Inhibition of Akt activation by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the presence of putative p63-binding sites in the pten promoter led us to investigate whether ΔNp63α regulates PTEN expression. Knockdown of ΔNp63α led to increases in PTEN levels and loss of activated Akt, while overexpression of ΔNp63α decreased PTEN levels and elevated active Akt. The repression of PTEN by ΔNp63α occurs independently of p53 status, as loss of ΔNp63α increases PTEN expression in cell lines with and without functional p53. In addition, decreased levels of ΔNp63α resulted in an increase in nuclear PTEN. Conversely, in vivo nuclear PTEN was absent in the proliferative basal layer of the epidermis where ΔNp63α expression is highest. Additionally, we show that in keratinocytes a balance between ΔNp63α and PTEN regulates Akt activation and maintains normal proliferation rates. This balance is disrupted in non-melanoma skin cancers through increased ΔNp63α levels, and could enhance proliferation and subsequent neoplastic development. Our studies show that ΔNp63α negatively regulates PTEN, thereby providing a feedback loop between PTEN, Akt and ΔNp63α, which has an integral role in skin cancer development.