Alessandra Pollice

Downregulation of ΔNp63α in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation

The tumor suppressor p14ARF inhibits cell growth in response to oncogenic stress in a p53¬dependent and independent manner. However, new physiologic roles for ARF activation have been proposed. We have previously demonstrated that ARF interacts with p63, influencing its transcriptional activity. p63 is a member of the p53 family involved in skin and limb development, as well as in the homeostasis of mature epidermis. Here, we show that, in human keratinocytes, as well as in tumor-derived cell lines, ARF targets ΔNp63α, the most abundantly expressed p63 isoform, to proteasomal degradation by stimulating its sumoylation. Interestingly, we have observed an increase of ARF expression in differentiating keratinocytes, that is concomitant to the already described upregulation of SUMO2/3. Remarkably, we found that ΔNp63α is preferentially sumoylated by SUMO2, instead of SUMO1, and p14ARF increases the efficiency of this process.

The promiscuity of ARF interactions with the proteasome.

The tumor suppressor ARF is one of the most important oncogenic stress sensors in mammalian cells. Its effect is exerted through the interaction with different cellular partners, often resulting in their functional inactivation. This review focuses on the role played by the proteasome in ARF regulation of protein turnover and the function of most of its interacting partners. Specific proteasome components appear to be involved in the regulation of ARF turnover, bringing to light a complex network of interactions between ARF and the proteasome.

Inhibition of p63 Transcriptional Activity by p14ARF: Functional and Physical Link between Human ARF Tumor Suppressor and a Member of the p53 Family

ABSTRACT The ARF/MDM2/p53 pathway is a principal defense mechanism to protect the organism from uncontrolled effects of deregulated oncogenes. Oncogenes activate ARF, which interacts with and inhibits the ubiquitin ligase MDM2, resulting in p53 stabilization and activation. Once stabilized and activated, p53 can either induce or repress a wide array of different gene targets, which in turn can regulate cell cycle, DNA repair, and a number of apoptosis-related genes. Here we show that, unlike p53, p63, a member of the p53 family, directly interacts with p14ARF. Through this interaction ARF inhibits p63-mediated transactivation and transrepression. In p63-transfected cells, ARF, which normally localizes into nucleoli, accumulates in the nucleoplasm. Based on these observations, we suggest that stimuli inducing p14ARF expression can, at the same time, activate p53 and impair p63 transcriptional activity, altering the pattern of p53 target gene expression. Here we show, for the first time, a physical and functional link between the p14ARF tumor suppressor protein and p63, a member of the p53 family.

TBP-1 protects the human oncosuppressor p14ARF from proteasomal degradation.

The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer. The mechanisms that regulate alternative reading frame (ARF) turnover have been obscure for long time, being ARF a relatively stable protein. Recently, it has been described that its degradation depends, at least in part, on the proteasome and that it can be subjected to N-terminal ubiquitination. We have previously reported that ARF protein levels are regulated by TBP-1 (Tat-Binding Protein 1), a multifunctional protein, component of the regulatory subunit of the proteasome, involved in different cellular processes. Here we demonstrate that the stabilization effect exerted by TBP-1 requires an intact N-terminal 39 amino acids in ARF and occurs independently from N-terminal ubiquitination of the protein. Furthermore, we observed that ARF can be degraded in vitro by the 20S proteasome, in the absence of ubiquitination and this effect can be counteracted by TBP-1. These observations seem relevant in the comprehension of the regulation of ARF metabolism as, among the plethora of cellular ARFs interactors already identified, only NPM/B23 and TBP-1 appear to be involved in the control of ARF intracellular levels.

The p63 Protein Isoform ΔNp63α Modulates Y-box Binding Protein 1 in Its Subcellular Distribution and Regulation of Cell Survival and Motility Genes

Background: YB-1 is a multifunctional protein that affects transcription, splicing, and translation. Results: ΔNp63α, the main p63 protein isoform, interacts with YB-1 and affects YB-1 subcellular localization and regulation of cell survival and motility genes. Conclusion: ΔNp63α and YB-1 interaction inhibits epithelial to mesenchymal transition and tumor cell motility. Significance: This is the first demonstration of a physical and functional interaction between YB-1 and ΔNp63α oncoproteins. The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.

The Hay Wells Syndrome-Derived TAp63αQ540L Mutant has Impaired Transcriptional and Cell Growth Regulatory Activity

P63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome, ADULT (acro, dermato, ungual, lacrimal, tooth) syndrome and AEC (ankyloblepharon, ectodermal dysplasia, clefting) syndrome (also called Hay-Wells syndrome). The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. In this paper we report a study on the TAp63α isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63α wt, δNp63α or the TAp63αQ540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63α and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.

A Biochemical and Cellular Approach to Explore the Antiproliferative and Prodifferentiative Activity of Aloe Arborescens Leaf Extract

Aloe arborescens Miller, belonging to the Aloe genus (Liliaceae family), is one of the main varieties of Aloe used worldwide. Although less characterized than the commonest Aloe vera, Aloe arborescens is known to be richer in beneficial phytotherapeutic, anticancer, and radio‐protective properties. It is commonly used as a pharmaceutical ingredient for its effect in burn treatment and ability to increase skin wound healing properties. However, very few studies have addressed the biological effects of Aloe at molecular level. The aim of the research is to provide evidences for the antiproliferative properties of Aloe arborescens crude leaf extract using an integrated proteomic and cellular biological approach. We analysed the composition of an Aloe arborescens leaf extract by gas chromatography‐mass spectrometry analysis. We found it rich in Aloe‐emodin, a hydroxylanthraquinone with known antitumoral activity and in several compounds with anti‐oxidant properties. Accordingly, we show that the Aloe extract has antiproliferative effects on several human transformed cell lines and exhibits prodifferentiative effects on both primary and immortalized human keratinocyte. Proteomic analysis of whole cell extracts revealed the presence of proteins with a strong antiproliferative and antimicrobial activity specifically induced in human keratinocytes by Aloe treatment supporting its application as a therapeutical agent. Copyright

Mimicking p14ARF Phosphorylation Influences Its Ability to Restrain Cell Proliferation

The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered loci in human cancer. It is generally accepted that ARF is involved in oncogenic checkpoint pathways by sensitizing incipient cancer cells to undergo growth arrest or apoptosis through both p53-dependent and independent pathways. While intensive studies have been focused on ARF activation at the transcriptional level, only recently mechanisms governing ARF turnover have been identified. Here, we show for the first time that p14ARF is a PKC target. Prediction analysis showed many potential phosphorylation sites in PKC consensus sequences within ARF protein, and, among them, the threonine at position 8 was the most conserved. Substitution of this threonine influences both ARF stability and localization. Furthermore, a phosphomimetic ARF mutation reduces the ability to arrest cell growth although the ability to bind MDM2 and stabilize p53 result unaffected. Thus we propose that phosphorylation of ARF in both immortalized and tumor cell lines could be a mechanism to escape ARF surveillance following proliferative and oncogenic stress.

Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and Growth

It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis.

A dominant mutation etiologic for human tricho‐dento‐osseous syndrome impairs the ability of DLX3 to downregulate ΔNp63α

The homeodomain transcription factors play crucial roles in many developmental processes ranging from organization of the body plan to differentiation of individual tissues. The homeodomain protein Distal‐less‐3 (DLX3) has an essential role in epidermal stratification and development of ectodermal appendages, placenta and bones. A four‐nucleotide deletion in the human DLX3 gene is etiologic for the human hereditary tricho‐dento‐osseous (TDO) ectodermal dysplasia, a dominant syndrome characterized by abnormalities in hair, nails, teeth, and bones. We have previously demonstrated that DLX3 gene expression induces degradation of ΔNp63α, a specific product of the TP63 gene, a master regulator of multi‐layered epithelia. Here we show that the DLX3TDO mutant protein is unable to promote ΔNp63α protein degradation and impairs the expression of cell cycle regulatory proteins and skin differentiation markers. However, we found that in cell expressing equal amounts of mutant and wild‐type DLX3, ΔNp63α protein level is efficiently regulated implying that genetic heterozygosity at the DLX3 locus protects TDO patients from developing severe p63‐associated skin defects. J. Cell. Physiol. 226: 2189–2197, 2011.