Ivano Amelio

Serine and glycine metabolism in cancer

Highlights • Serine and glycine are essential metabolites for cancer cells.• Serine and glycine provide precursors for macromolecules and antioxidant defence.• Metabolic enzymes of serine and glycine biosynthesis are upregulated in cancer.• Innovative anticancer therapy is aiming to target serine and glycine biosynthesis.

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration.

A differentiation-promoting micro-RNA regulates actin cable dynamics, intercellular adhesion, and cell migration in human and mouse epidermis.

p73 regulates serine biosynthesis in cancer

Activation of serine biosynthesis supports growth and proliferation of cancer cells. Human cancers often exhibit overexpression of phosphoglycerate dehydrogenase (PHGDH), the metabolic enzyme that catalyses the reaction that diverts serine biosynthesis from the glycolytic pathway. By refueling serine biosynthetic pathways, cancer cells sustain their metabolic requirements, promoting macromolecule synthesis, anaplerotic flux and ATP. Serine biosynthesis intersects glutaminolysis and together with this pathway provides substrates for production of antioxidant GSH. In human lung adenocarcinomas we identified a correlation between serine biosynthetic pathway and p73 expression. Metabolic profiling of human cancer cell line revealed that TAp73 activates serine biosynthesis, resulting in increased intracellular levels of serine and glycine, associated to accumulation of glutamate, tricarboxylic acid (TCA) anaplerotic intermediates and GSH. However, at molecular level p73 does not directly regulate serine metabolic enzymes, but transcriptionally controls a key enzyme of glutaminolysis, glutaminase-2 (GLS-2). p73, through GLS-2, favors conversion of glutamine in glutamate, which in turn drives the serine biosynthetic pathway. Serine and glutamate can be then employed for GSH synthesis, thus the p73-dependent metabolic switch enables potential response against oxidative stress. In knockdown experiment, indeed, TAp73 depletion completely abrogates cancer cell proliferation capacity in serine/glycine-deprivation, supporting the role of p73 to help cancer cells under metabolic stress. These findings implicate p73 in regulation of cancer metabolism and suggest that TAp73 influences glutamine and serine metabolism, affecting GSH synthesis and determining cancer pathogenesis.

High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy

Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine—a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.

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.

TAp73 opposes tumor angiogenesis by promoting hypoxia-inducible factor 1α degradation

Significance Adaptation to hypoxia promotes cancer progression, resulting in enhanced patient mortality. Activation of hypoxia-inducible factor 1 (HIF-1) leads to a transcriptional switch, which, regulating angiogenesis, metabolism, and survival, results in hypoxia adaptation. In cancer, increased HIF-1 levels can be a result of either intratumoral hypoxia or the altered function of tumor suppressors. Our study demonstrates that the tumor suppressor TAp73, a member of the p53 family of genes, opposes HIF-1 activation in cancer cells, resulting in reduced angiogenesis and tumor progression. TAp73-depleted mice show increased tumorigenicity, associated with increased HIF-1 signaling and angiogenesis. Expression of TAp73 in human cancers predicts good survival outcome and retrocorrelates with HIF-1 expression and activation. The TAp73/HIF-1 axis plays a critical role in cancer pathogenesis. Tumor hypoxia and hypoxia-inducible factor 1 (HIF-1) activation are associated with cancer progression. Here, we demonstrate that the transcription factor TAp73 opposes HIF-1 activity through a nontranscriptional mechanism, thus affecting tumor angiogenesis. TAp73-deficient mice have an increased incidence of spontaneous and chemically induced tumors that also display enhanced vascularization. Mechanistically, TAp73 interacts with the regulatory subunit (α) of HIF-1 and recruits mouse double minute 2 homolog into the protein complex, thus promoting HIF-1α polyubiquitination and consequent proteasomal degradation in an oxygen-independent manner. In human lung cancer datasets, TAp73 strongly predicts good patient prognosis, and its expression is associated with low HIF-1 activation and angiogenesis. Our findings, supported by in vivo and clinical evidence, demonstrate a mechanism for oxygen-independent HIF-1 regulation, which has important implications for individualizing therapies in patients with cancer.

MicroRNAs and p63 in epithelial stemness

MicroRNAs (miRs) are a class of small noncoding RNAs that suppress the expression of protein-coding genes by repressing protein translation. Although the roles that miRs and the miR processing machinery have in regulating epithelial stem cell biology are not fully understood, their fundamental contributions to these processes have been demonstrated over the last few years. The p53-family member p63 is an essential transcription factor for epidermal morphogenesis and homeostasis. p63 functions as a determinant for keratinocyte cell fate and helps to regulate the balance between stemness, differentiation and senescence. An important factor that regulates p63 function is the reciprocal interaction between p63 and miRs. Some miRs control p63 expression, and p63 regulates the miR expression profile in the epidermis. p63 controls miR expression at different levels. It directly regulates the transcription of several miRs and indirectly regulates their processing by regulating the expression of the miR processing components Dicer and DGCR8. In this review, we will discuss the recent findings on the miR–p63 interaction in epidermal biology, particularly focusing on the ΔNp63-dependent regulation of DGCR8 recently described in the ΔNp63−/− mouse. We provide a unified view of the current knowledge and discuss the apparent discrepancies and perspective therapeutic opportunities.

TAp73 is required for spermatogenesis and the maintenance of male fertility

Significance Defects in spermatogenesis, many of which are unexplained, underlie the infertility problems of ∼20% of couples. Although specific roles for the p53 family members in female fertility have been described, their involvement in spermatogenesis is largely unexpected. Using gene-targeted mice, we have demonstrated that deficiency of TAp73, but not p53 or ∆Np73, leads to male infertility caused by severely impaired germ cell differentiation and maturation to viable sperms in the testes. Importantly, our work has established that TAp73, but not p53, regulates many genes involved in spermatogenesis. Thus, our results provide previously unidentified in vivo evidence that TAp73 is a “guardian” of male germ cells and may point toward a novel avenue for the diagnosis and management of male infertility. The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility.

DRUGSURV: a resource for repositioning of approved and experimental drugs in oncology based on patient survival information

The use of existing drugs for new therapeutic applications, commonly referred to as drug repositioning, is a way for fast and cost-efficient drug discovery. Drug repositioning in oncology is commonly initiated by in vitro experimental evidence that a drug exhibits anticancer cytotoxicity. Any independent verification that the observed effects in vitro may be valid in a clinical setting, and that the drug could potentially affect patient survival in vivo is of paramount importance. Despite considerable recent efforts in computational drug repositioning, none of the studies have considered patient survival information in modelling the potential of existing/new drugs in the management of cancer. Therefore, we have developed DRUGSURV; this is the first computational tool to estimate the potential effects of a drug using patient survival information derived from clinical cancer expression data sets. DRUGSURV provides statistical evidence that a drug can affect survival outcome in particular clinical conditions to justify further investigation of the drug anticancer potential and to guide clinical trial design. DRUGSURV covers both approved drugs (∼1700) as well as experimental drugs (∼5000) and is freely available at http://www.bioprofiling.de/drugsurv.

p63 the guardian of human reproduction

p63 is a transcriptional factor implicated in cancer and development. The presence in TP63 gene of alternative promoters allows expression of one isoform containing the N-terminal transactivation domain (TA isoform) and one N-terminal truncated isoform (ΔN isoform). Complete ablation of all p63 isoforms produced mice with fatal developmental abnormalities, including lack of epidermal barrier, limbs and other epidermal appendages. Specific TAp63-null mice, although they developed normally, failed to undergo in DNA damage-induced apoptosis during primordial follicle meiotic arrest, suggesting a p63 involvement in maternal reproduction. Recent findings have elucidated the role in DNA damage response of a novel Hominidae p63 isoform, GTAp63, specifically expressed in human spermatic precursors. Thus, these findings suggest a unique strategy of p63 gene, to evolve in order to preserve the species as a guardian of reproduction. Elucidation of the biological basis of p63 function in reproduction may provide novel approaches to the control of human fertility.