Maria Vivo

The Human Tumor Suppressor ARF Interacts with Spinophilin/Neurabin II, a Type 1 Protein-phosphatase-binding Protein

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p14ARF (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.

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.

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.

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.

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.

MDM2-Mediated Degradation of p14ARF: A Novel Mechanism to Control ARF Levels in Cancer Cells

We here show a new relationship between the human p14ARF oncosuppressor and the MDM2 oncoprotein. MDM2 overexpression in various cancer cell lines causes p14ARF reduction inducing its degradation through the proteasome. The effect does not require the ubiquitin ligase activity of MDM2 and preferentially occurs in the cytoplasm. Interestingly, treatment with inhibitors of the PKC (Protein Kinase C) pathway and use of p14ARF phosphorylation mutants indicate that ARF phosphorylation could play a role in MDM2 mediated ARF degradation reinforcing our previous observations that ARF phosphorylation influences its stability and biological activity. Our study uncovers a new potentially important mechanism through which ARF and MDM2 can counterbalance each other during the tumorigenic process.

Y-box Binding Protein-1 Is Part of a Complex Molecular Network Linking ΔNp63α to the PI3K/akt Pathway in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene‐activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y‐box binding 1 (YB‐1 or YBX1) oncoproteins. Here, we show that knockdown of YB‐1 in spontaneously immortalized HaCaT and non‐metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB‐1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB‐1 depletion and promotes cell survival. In summary, our results unveil a functional cross‐talk between YB‐1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells. J. Cell. Physiol. 230: 2067–2074, 2015.

p14ARF interacts with the focal adhesion kinase and protects cells from anoikis

The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.