ΔNp63α controls YB-1 protein stability: evidence on YB-1 as a new player in keratinocyte differentiation.

Y-box binding protein 1 (YBX-1 or YB-1) is an oncoprotein that promotes replicative immortality, tumor cell invasion and metastasis. The increase in the abundance of YB-1 in the cell or YB-1 translocation from the cytoplasm to the nucleus is characteristic of malignant cell growth. We have previously reported that ΔNp63α, a transcription factor that is known to play a pivotal role in keratinocyte proliferation and differentiation, promotes YB-1 nuclear accumulation. Here, we show that YB-1 is highly expressed in proliferating keratinocytes and is down-regulated during keratinocyte differentiation. ΔNp63α reduces YB-1 protein turnover and leads to accumulation of ubiquitin-conjugated YB-1 into the nucleus. Reduction of YB-1 protein level, following treatment with a DNA-damaging agent, is inhibited by ΔNp63α suggesting that YB-1 and ΔNp63α interplay can support keratinocyte proliferation and protect cells from apoptosis under genotoxic stress.

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.

The p63 protein isoform ΔNp63α modulates Y-box binding protein 1 in its subcellular distribution and regulation of cell survival and motility genes.

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.

MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation.

Tight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the DeltaNp63alpha protein. We found that MDM2 binds DeltaNp63alpha in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for DeltaNp63alpha nuclear export and subsequent degradation, whereas the MDM2 ring-finger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of DeltaNp63alpha by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the alpha and beta tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous DeltaNp63alpha in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative DeltaNp63alpha protein.

Mutation of the Conserved Threonine 8 within the Human ARF Tumour Suppressor Protein Regulates Autophagy.

BACKGROUND: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. METHODS: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. RESULTS: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells' inability to elicit autophagosomes formation upon T8D expression. CONCLUSIONS: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein's ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

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 DLX3(TDO) 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.

Pancreatic Progenitor Commitment Is Marked by an Increase in Ink4a/Arf Expression.

The identification of the molecular mechanisms controlling early cell fate decisions in mammals is of paramount importance as the ability to determine specific lineage differentiation represents a significant opportunity for new therapies. Pancreatic Progenitor Cells (PPCs) constitute a regenerative reserve essential for the maintenance and regeneration of the pancreas. Besides, PPCs represent an excellent model for understanding pathological pancreatic cellular remodeling. Given the lack of valid markers of early endoderm, the identification of new ones is of fundamental importance. Both products of the Ink4a/Arf locus, in addition to being critical cell-cycle regulators, appear to be involved in several disease pathologies. Moreover, the locus' expression is epigenetically regulated in ES reprogramming processes, thus constituting the ideal candidates to modulate PPCs homeostasis. In this study, starting from mouse embryonic stem cells (mESCs), we analyzed the early stages of pancreatic commitment. By inducing mESCs commitment to the pancreatic lineage, we observed that both products of the Cdkn2a locus, Ink4a and Arf, mark a naïve pancreatic cellular state that resembled PPC-like specification. Treatment with epi-drugs suggests a role for chromatin remodeling in the CDKN2a (Cycline Dependent Kinase Inhibitor 2A) locus regulation in line with previous observations in other cellular systems. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

Identification of DeltaNp63alpha protein interactions by mass spectrometry.

p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (DeltaN) a transactivation domain. DeltaNp63alpha is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of DeltaNp63alpha, we set out to identify DeltaNp63alpha interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential DeltaNp63alpha binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of DeltaNp63alpha with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that DeltaNp63alpha exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of DeltaNp63alpha in mRNA metabolism and transport.

Dual Role of the Alternative Reading Frame ARF Protein in Cancer.

The CDKN2a/ARF locus expresses two partially overlapping transcripts that encode two distinct proteins, namely p14ARF (p19Arf in mouse) and p16INK4a, which present no sequence identity. Initial data obtained in mice showed that both proteins are potent tumor suppressors. In line with a tumor-suppressive role, ARF-deficient mice develop lymphomas, sarcomas, and adenocarcinomas, with a median survival rate of one year of age. In humans, the importance of ARF inactivation in cancer is less clear whereas a more obvious role has been documented for p16INK4a. Indeed, many alterations in human tumors result in the elimination of the entire locus, while the majority of point mutations affect p16INK4a. Nevertheless, specific mutations of p14ARF have been described in different types of human cancers such as colorectal and gastric carcinomas, melanoma and glioblastoma. The activity of the tumor suppressor ARF has been shown to rely on both p53-dependent and independent functions. However, novel data collected in the last years has challenged the traditional and established role of this protein as a tumor suppressor. In particular, tumors retaining ARF expression evolve to metastatic and invasive phenotypes and in humans are associated with a poor prognosis. In this review, the recent evidence and the molecular mechanisms of a novel role played by ARF will be presented and discussed, both in pathological and physiological contexts.

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.

Sumoylation and ubiquitylation crosstalk in the control of ΔNp63α protein stability.

ΔNp63α is finely and strictly regulated during embryogenesis and differentiation. ΔNp63α is the only p63 isoform degraded by the proteasome after Ubiquitin and SUMO (Small Ubiquitin-like MOdifier) conjugation. Here, we show that p63 ubiquitylation per se is not the signal triggering p63 proteasomal degradation. Taking advantage of natural ΔNp63α mutants isolated by patients with Split Hand and Foot Malformation IV syndrome, we found that SUMO and Ub modifications are not redundant and both are required to guarantee efficient ΔNp63α degradation. Here, we present evidence that sumoylation and ubiquitylation of ΔNp63α are strongly intertwined, and none of the two can efficiently occur if the other is impaired.

PKC Dependent p14ARF Phosphorylation on Threonine 8 Drives Cell Proliferation.

ARF role as tumor suppressor has been challenged in the last years by several findings of different groups ultimately showing that its functions can be strictly context dependent. We previously showed that ARF loss in HeLa cells induces spreading defects, evident as rounded morphology of depleted cells, accompanied by a decrease of phosphorylated Focal Adhesion Kinase (FAK) protein levels and anoikis. These data, together with previous finding that a PKC dependent signalling pathway can lead to ARF stabilization, led us to the hypothesis that ARF functions in cell proliferation might be regulated by phosphorylation. In line with this, we show here that upon spreading ARF is induced through PKC activation. A constitutive-phosphorylated ARF mutant on the conserved threonine 8 (T8D) is able to mediate both cell spreading and FAK activation. Finally, ARF-T8D expression confers growth advantage to cells thus leading to the intriguing hypothesis that ARF phosphorylation could be a mechanism through which pro-proliferative or anti proliferative signals could be transduced inside the cells in both physiological and pathological conditions.

Oxidative Stress Causes Enhanced Secretion of YB-1 Protein that Restrains Proliferation of Receiving Cells.

The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein that regulates a variety of fundamental biological processes including cell proliferation and migration, DNA damage, matrix protein synthesis and chemotaxis. The plethora of functions assigned to YB-1 is strictly dependent on its subcellular localization. In resting cells, YB-1 localizes to cytoplasm where it is a component of messenger ribonucleoprotein particles. Under stress conditions, YB-1 contributes to the formation of stress granules (SGs), cytoplasmic foci where untranslated messenger RNAs (mRNAs) are sorted or processed for reinitiation, degradation, or packaging into ribonucleoprotein particles (mRNPs). Following DNA damage, YB-1 translocates to the nucleus and participates in DNA repair thereby enhancing cell survival. Recent data show that YB-1 can also be secreted and YB-1-derived polypeptides are found in plasma of patients with sepsis and malignancies. Here we show that in response to oxidative insults, YB-1 assembly in SGs is associated with an enhancement of YB-1 protein secretion. An enriched fraction of extracellular YB-1 (exYB-1) significantly inhibited proliferation of receiving cells and such inhibition was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of Np63 protein level. All together, these data show that acute oxidative stress causes sustained release of YB-1 as a paracrine/autocrine signal that stimulate cell cycle arrest.

Inhibition of p63 transcriptional activity by p14ARF: functional and physical link between human ARF tumor suppressor and a member of the p53 family.

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 p14(ARF). 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 p14(ARF) 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 p14(ARF) tumor suppressor protein and p63, a member of the p53 family.

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.

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 regulatory mechanism involving TBP-1/Tat-Binding Protein 1 and Akt/PKB in the control of cell proliferation.

TBP-1 /Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a multifunctional protein, originally identified as a regulator of HIV-1-Tat mediated transcription. It is an AAA-ATPase component of the 19S regulative subunit of the proteasome and, as other members of this protein family, fulfils different cellular functions including proteolysis and transcriptional regulation. We and others reported that over expression of TBP-1 diminishes cell proliferation in different cellular contexts with mechanisms yet to be defined. Accordingly, we demonstrated that TBP-1 binds to and stabilizes the p14ARF oncosuppressor increasing its anti-oncogenic functions. However, TBP-1 restrains cell proliferation also in the absence of ARF, raising the question of what are the molecular pathways involved. Herein we demonstrate that stable knock-down of TBP-1 in human immortalized fibroblasts increases cell proliferation, migration and resistance to apoptosis induced by serum deprivation. We observe that TBP-1 silencing causes activation of the Akt/PKB kinase and that in turn TBP-1, itself, is a downstream target of Akt/PKB. Moreover, MDM2, a known Akt target, plays a major role in this regulation. Altogether, our data suggest the existence of a negative feedback loop involving Akt/PKB that might act as a sensor to modulate TBP-1 levels in proliferating cells.

Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation.

The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant DeltaNp63alpha in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of DeltaNp63alpha are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raf1 phosphorylation. Dlx3 is unable to promote p63 degradation in Raf1 depleted MEF cells or upon pharmacological knockdown of Raf1. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of DeltaNp63alpha protein level, a mechanism that may contribute to reduce the abundance of DeltaNp63alpha during differentiation of stratified epithelia.

Downregulation of DeltaNp63alpha in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation.

The tumor suppressor p14(ARF) 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 DeltaNp63alpha, 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 DeltaNp63alpha is preferentially sumoylated by SUMO2, instead of SUMO1, and p14(ARF) increases the efficiency of this process.

Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias.

Ectodermal dysplasias (EDs) are a group of human pathological conditions characterized by anomalies in organs derived from epithelial-mesenchymal interactions during development. Dlx3 and p63 act as part of the transcriptional regulatory pathways relevant in ectoderm derivatives, and autosomal mutations in either of these genes are associated with human EDs. However, the functional relationship between both proteins is unknown. Here, we demonstrate that Dlx3 is a downstream target of p63. Moreover, we show that transcription of Dlx3 is abrogated by mutations in the sterile alpha-motif (SAM) domain of p63 that are associated with ankyloblepharon-ectodermal dysplasia-clefting (AEC) dysplasias, but not by mutations found in ectrodactylyectodermal dysplasia-cleft lip/palate (EEC), Limb-mammary syndrome (LMS) and split hand-foot malformation (SHFM) dysplasias. Our results unravel aspects of the transcriptional cascade of events that contribute to ectoderm development and pathogenesis associated with p63 mutations.