Angelo Peschiaroli

Itch: A HECT-type E3 ligase regulating immunity, skin and cancer

The HECT-type E3 ubiquitin ligase (E3) Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease, including lung and stomach inflammation and hyperplasia of lymphoid and hematopoietic cells. The involvement of Itch in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review aims to bring together a growing body of work exploring Itch-regulated biological processes, and to highlight recent discoveries on the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Our contribution is also an endeavor to correlate Itch substrate specificity with the pathological defects manifested by the mutant Itchy mice.

p63 Sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling.

Significance p63, the sister homolog of p53, is a master regulator of epithelial stem cell (SC) biology. p63 is indeed intimately implicated in the maintenance of the self-renewal capacity of stratified epithelia and their derivatives, including the mammary gland. Although the physiological role of p63 in normal mammary SCs is now acknowledged, proof of its implications in breast cancer SCs remains elusive. Here, we find that mammary cancer stem cells (CSCs) possess increased levels of p63 expression compared with normal progenitors. p63 promotes self-renewal and expansion of mammary CSCs and breast tumor growth in vivo. Additionally, this study provides a link between p63 and the Sonic Hedgehog signaling pathway in the regulation of breast cancer stemness. The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog (Shh), GLI family zinc finger 2 (Gli2), and Patched1 (Ptch1) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway.

The F-box protein FBXO45 promotes the proteasome-dependent degradation of p73

The transcription factor p73, a member of the p53 family, mediates cell-cycle arrest and apoptosis in response to DNA damage-induced cellular stress, acting thus as a proapoptotic gene. Similar to p53, p73 activity is regulated by post-translational modification, including phosphorylation, acetylation and ubiquitylation. In C. elegans, the F-box protein FSN-1 controls germline apoptosis by regulating CEP-1, the single ancestral p53 family member. Here we report that FBXO45, the human ortholog of FSN-1, binds specifically to p73 triggering its proteasome-dependent degradation. Importantly, SCFFBXO45 ubiquitylates p73 both in vivo and in vitro. Moreover, siRNA-mediated depletion of FBXO45 stabilizes p73 and concomitantly induces cell death in a p53-independent manner. All together, these results show that the orphan F-box protein FBXO45 regulates the stability of p73, highlighting a conserved pathway evolved from nematode to human by which the p53 members are regulated by an SCF-dependent mechanism.

p63 regulates glutaminase 2 expression.

The transcription factor p63 is critical for many biological processes, including development and maintenance of epidermal tissues and tumorigenesis. Here, we report that the TAp63 isoforms regulate cell metabolism through the induction of the mitochondrial glutaminase 2 (GLS2) gene both in primary cells and tumor cell lines. By ChIP analysis and luciferase assay, we confirmed that TAp63 binds directly to the p53/p63 consensus DNA binding sequence within the GLS2 promoter region. Given the critical role of p63 in epidermal differentiation, we have investigated the regulation of GLS2 expression during this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes.

GLS2 is transcriptionally regulated by p73 and contributes to neuronal differentiation

The amino acid Glutamine is converted into Glutamate by a deamidation reaction catalyzed by the enzyme Glutaminase (GLS). Two isoforms of this enzyme have been described, and the GLS2 isoform is regulated by the tumor suppressor gene p53. Here, we show that the p53 family member TAp73 also drives the expression of GLS2. Specifically, we demonstrate that TAp73 regulates GLS2 during retinoic acid-induced terminal neuronal differentiation of neuroblastoma cells, and overexpression or inhibition of GLS2 modulates neuronal differentiation and intracellular levels of ATP. Moreover, inhibition of GLS activity, by removing Glutamine from the growth medium, impairs in vitro differentiation of cortical neurons. Finally, expression of GLS2 increases during mouse cerebellar development. Although, p73 is dispensable for the in vivo expression of GLS2, TAp73 loss affects GABA and Glutamate levels in cortical neurons. Together, these findings suggest a role for GLS2 acting, at least in part, downstream of p73 in neuronal differentiation and highlight a possible role of p73 in regulating neurotransmitter synthesis.

The ubiquitin-specific protease USP47 is a novel beta-TRCP interactor regulating cell survival

Ubiquitin-specific proteases (USPs) are a subclass of cysteine proteases that catalyze the removal of ubiquitin (either monomeric or chains) from substrates, thus counteracting the activity of E3 ubiquitin ligases. Although the importance of USPs in a multitude of processes, from hereditary cancer to neurodegeneration, is well established, our knowledge on their mode of regulation, substrate specificity and biological function is quite limited. In this study we identify USP47 as a novel interactor of the E3 ubiquitin ligase, Skp1/Cul1/F-box protein β-transducin repeat-containing protein (SCFβ-Trcp). We found that both β-Trcp1 and β-Trcp2 bind specifically to USP47, and point mutations in the β-Trcp WD-repeat region completely abolished USP47 binding, indicating an E3-substrate-type interaction. However, unlike canonical β-Trcp substrates, USP47 protein levels were neither affected by silencing of β-Trcp nor modulated in a variety of processes, such as cell-cycle progression, DNA damage checkpoint responses or tumor necrosis factor (TNF) pathway activation. Notably, genetic or siRNA-mediated depletion of USP47 induced accumulation of Cdc25A, decreased cell survival and augmented the cytotoxic effects of anticancer drugs. In conclusion, we showed that USP47, a novel β-Trcp interactor, regulates cell growth and survival, potentially providing a novel target for anticancer therapies.

Itch self-polyubiquitylation occurs through lysine-63 linkages.

Itch, an E3 protein ubiquitin ligase (E3), which belongs to the homologous to E6-AP carboxy terminus (HECT)-type subfamily, catalyzes its own ubiquitylation. The precise nature of Itch-mediated self-modification and its biological outcome are not completely understood. Here, we show that Itch auto-ubiquitylation is an intermolecular reaction generating Lys63-linkages, rather than the Lys48-linked polyubiquitin chains that target proteins for proteasomal degradation. As a result, Itch is a relatively high stable protein, whose levels are not significantly affected by treatment by either proteasome or lysosome inhibitors. Furthermore, we demonstrate that the decay rate of a catalytic inactive Itch mutant, which is devoided of self-ubiquitylating activity, is barely indistinguishable from the one of the wild-type protein. These data definitely establish a nondegradative role for Lys63-linked Itch self-ubiquitylation.

The E3 ubiquitin ligase WWP1 regulates ΔNp63-dependent transcription through Lys63 linkages

The transcription factor p63, a member of the p53 family, plays a crucial role in epithelial development and tumorigenesis through the regulation of epithelial progenitor cell proliferation, differentiation and apoptosis. Similarly to p53, p63 activity is regulated by post-translational modifications, including ubiquitylation. Here, we report that the WWP1 E3 ubiquitin ligase binds specifically to ΔNp63 isoform but it does not trigger ΔNp63 proteasome-dependent degradation. Accordingly, we found that WWP1-dependent ubiquitylation of ΔNp63 occurs through the formation of Lys63-linked poly-ubiquitin chains. Importantly, we found that WWP1 is able to increase ΔNp63-dependent transcription and depletion of WWP1 in human primary keratinocytes induces cell cycle arrest. All together these results indicate that WWP1 regulates ΔNp63 transcriptional activity, acting thus as a potential regulator of the proliferation and survival of epithelial-derived cells.

p63 controls cell migration and invasion by transcriptional regulation of MTSS1

Metastasis is a multistep cell-biological process, which is orchestrated by many factors, including metastasis activators and suppressors. Metastasis Suppressor 1 (MTSS1) was originally identified as a metastasis suppressor protein whose expression is lost in metastatic bladder and prostate carcinomas. However, recent findings indicate that MTSS1 acts as oncogene and pro-migratory factor in melanoma tumors. Here, we identify and characterized a molecular mechanism controlling MTSS1 expression, which impinges on a pro-tumorigenic role of MTSS1 in breast tumors. We found that in normal and in cancer cell lines ΔNp63 is able to drive the expression of MTSS1 by binding to a p63-binding responsive element localized in the MTSS1 locus. We reported that ΔNp63 is able to drive the migration of breast tumor cells by inducing the expression of MTSS1. Notably, in three human breast tumors data sets the MTSS1/p63 co-expression is a negative prognostic factor on patient survival, suggesting that the MTSS1/p63 axis might be functionally important to regulate breast tumor progression.

The Cul4A–DDB1 E3 ubiquitin ligase complex represses p73 transcriptional activity

The Cullin4A (cul4A)-dependent ligase (CDL4A) E3 has been implicated in a variety of biological processes, including cell cycle progression and DNA damage response. Remarkably, CDL4A exerts its function through both proteolytic and non-proteolytic events. Here, we show that the p53 family member p73 is able to interact with the CDL4A complex through its direct binding to the receptor subunit DNA-binding protein 1 (DDB1). As a result, the CDL4A complex is able to monoubiquitylate p73. Modification of p73 by CDL4A-mediated ubiquitylation does not affect p73 protein stability, but negatively regulates p73-dependent transcriptional activity. Indeed, genetic or RNA interference-mediated depletion of DDB1 induces the expression of several p73 target genes in a p53-independent manner. In addition, by exploiting a bioinformatic approach, we found that elevated expression of Cul4A in human breast carcinomas is associated with repression of p73 target genes. In conclusion, our findings add a novel insight into the regulation of p73 by the CDL4A complex, through the inhibition of its transcriptional function.