Hyaluronic acid metabolism and chemotherapy resistance: recent advances and therapeutic potential.

Hyaluronic acid (HA) is a major component of the extracellular matrix, providing essential mechanical scaffolding for cells and, at the same time, mediating essential biochemical signals required for tissue homeostasis. Many solid tumors are characterized by dysregulated HA metabolism, resulting in increased HA levels in cancer tissues. HA interacts with several cell surface receptors, such as cluster of differentiation 44 and receptor for hyaluronan-mediated motility, thus co-regulating important signaling pathways in cancer development and progression. In this review, we describe the enzymes controlling HA metabolism and its intracellular effectors emphasizing their impact on cancer chemotherapy resistance. We will also explore the current and future prospects of HA-based therapy, highlighting the opportunities and challenges in the field.

The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation.

The transcription factor ΔNp63 regulates epithelial stem cell function and maintains the integrity of stratified epithelial tissues by acting as transcriptional repressor or activator towards a distinct subset of protein-coding genes and microRNAs. However, our knowledge of the functional link between ∆Np63 transcriptional activity and long non-coding RNAs (lncRNAs) expression is quite limited. Here, we show that in proliferating human keratinocytes ∆Np63 represses the expression of the lncRNA NEAT1 by recruiting the histone deacetylase HDAC1 to the proximal promoter of NEAT1 genomic locus. Upon induction of differentiation, ∆Np63 down-regulation is associated by a marked increase of NEAT1 RNA levels, resulting in an increased assembly of paraspeckles foci both in vitro and in human skin tissues. RNA-seq analysis associated with global DNA binding profile (ChIRP-seq) revealed that NEAT1 associates with the promoter of key epithelial transcription factors sustaining their expression during epidermal differentiation. These molecular events might explain the inability of NEAT1-depleted keratinocytes to undergo the proper formation of epidermal layers. Collectively, these data uncover the lncRNA NEAT1 as an additional player of the intricate network orchestrating epidermal morphogenesis.

Senataxin and R-loops homeostasis: multifaced implications in carcinogenesis.

R-loops are inherent byproducts of transcription consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. These structures are of key importance in controlling numerous physiological processes and their homeostasis is tightly controlled by the activities of several enzymes deputed to process R-loops and prevent their unproper accumulation. Senataxin (SETX) is an RNA/DNA helicase which catalyzes the unwinding of RNA:DNA hybrid portion of the R-loops, promoting thus their resolution. The key importance of SETX in R-loops homeostasis and its relevance with pathophysiological events is highlighted by the evidence that gain or loss of function SETX mutations underlie the pathogenesis of two distinct neurological disorders. Here, we aim to describe the potential impact of SETX on tumor onset and progression, trying to emphasize how dysregulation of this enzyme observed in human tumors might impact tumorigenesis. To this aim, we will describe the functional relevance of SETX in regulating gene expression, genome integrity, and inflammation response and discuss how cancer-associated SETX mutations might affect these pathways, contributing thus to tumor development.

ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes.

SignificanceΔNp63 is a master regulator of skin homeostasis since it finely controls keratinocyte differentiation and proliferation. Here, we provide cellular and molecular evidence demonstrating the functional role of a ΔNp63 interactor, the R-loop-resolving enzyme Senataxin (SETX), in fine-tuning keratinocyte differentiation. We found that SETX physically binds the p63 DNA-binding motif present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus allowing efficient transcriptional termination and gene expression. These molecular events translate into the inability of SETX-depleted keratinocytes to undergo the correct epidermal differentiation program. Remarkably, SETX is dysregulated in cutaneous squamous cell carcinoma, suggesting its potential involvement in the pathogenesis of skin disorders.

Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation.

Hereditary Spastic Paraplegia (HSP) is a neurodegenerative disease most commonly caused by autosomal dominant mutations in the SPG4 gene encoding the microtubule-severing protein spastin. We hypothesise that SPG4-HSP is attributable to reduced spastin function because of haploinsufficiency; thus, therapeutic approaches which elevate levels of the wild-type spastin allele may be an effective therapy. However, until now, how spastin levels are regulated is largely unknown. Here, we show that the kinase HIPK2 regulates spastin protein levels in proliferating cells, in differentiated neurons and in vivo. Our work reveals that HIPK2-mediated phosphorylation of spastin at S268 inhibits spastin K48-poly-ubiquitination at K554 and prevents its neddylation-dependent proteasomal degradation. In a spastin RNAi neuronal cell model, overexpression of HIPK2, or inhibition of neddylation, restores spastin levels and rescues neurite defects. Notably, we demonstrate that spastin levels can be restored pharmacologically by inhibiting its neddylation-mediated degradation in neurons derived from a spastin mouse model of HSP and in patient-derived cells, thus revealing novel therapeutic targets for the treatment of SPG4-HSP.

The Impact of the Ubiquitin System in the Pathogenesis of Squamous Cell Carcinomas.

The ubiquitin system is a dynamic regulatory pathway controlling the activity, subcellular localization and stability of a myriad of cellular proteins, which in turn affects cellular homeostasis through the regulation of a variety of signaling cascades. Aberrant activity of key components of the ubiquitin system has been functionally linked with numerous human diseases including the initiation and progression of human tumors. In this review, we will contextualize the importance of the two main components of the ubiquitin system, the E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs), in the etiology of squamous cell carcinomas (SCCs). We will discuss the signaling pathways regulated by these enzymes, emphasizing the genetic and molecular determinants underlying their deregulation in SCCs.

HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3ß kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3ß at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1-dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3ß and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy.

Correction to: HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A novel oral micellar fenretinide formulation with enhanced bioavailability and antitumour activity against multiple tumours from cancer stem cells.

BACKGROUND: An increasing number of anticancer agents has been proposed in recent years with the attempt to overcome treatment-resistant cancer cells and particularly cancer stem cells (CSC), the major culprits for tumour resistance and recurrence. However, a huge obstacle to treatment success is the ineffective delivery of drugs within the tumour environment due to limited solubility, short circulation time or inconsistent stability of compounds that, together with concomitant dose-limiting systemic toxicity, contribute to hamper the achievement of therapeutic drug concentrations. The synthetic retinoid Fenretinide (4-hydroxy (phenyl)retinamide; 4-HPR) formerly emerged as a promising anticancer agent based on pre-clinical and clinical studies. However, a major limitation of fenretinide is traditionally represented by its poor aqueous solubility/bioavailability due to its hydrophobic nature, that undermined the clinical success of previous clinical trials. METHODS: Here, we developed a novel nano-micellar fenretinide formulation called bionanofenretinide (Bio-nFeR), based on drug encapsulation in an ion-pair stabilized lipid matrix, with the aim to raise fenretinide bioavailability and antitumour efficacy. RESULTS: Bio-nFeR displayed marked antitumour activity against lung, colon and melanoma CSC both in vitro and in tumour xenografts, in absence of mice toxicity. Bio-nFeR is suitable for oral administration, reaching therapeutic concentrations within tumours and an unprecedented therapeutic activity in vivo as single agent. CONCLUSION: Altogether, our results indicate Bio-nFeR as a novel anticancer agent with low toxicity and high activity against tumourigenic cells, potentially useful for the treatment of solid tumours of multiple origin.

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of ßTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated ßTrCP, the substrate-receptor subunit of the SCFßTrCP ubiquitin ligase, and promotes ßTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors.

p63 at the Crossroads between Stemness and Metastasis in Breast Cancer.

After lung cancer, breast cancer (BC) is the most frequent cause of cancer death among women, worldwide. Although advances in screening approaches and targeted therapeutic agents have decreased BC incidence and mortality, over the past five years, triple-negative breast cancer (TNBC) remains the breast cancer subtype that displays the worst prognosis, mainly due to the lack of clinically actionable targets. Genetic and molecular profiling has unveiled the high intrinsic heterogeneity of TNBC, with the basal-like molecular subtypes representing the most diffuse TNBC subtypes, characterized by the expression of basal epithelial markers, such as the transcription factor p63. In this review, we will provide a broad picture on the physiological role of p63, in maintaining the basal epithelial identity, as well as its involvement in breast cancer progression, emphasizing its relevance in tumor cell invasion and stemness.

ΔNp63 in squamous cell carcinoma: defining the oncogenic routes affecting epigenetic landscape and tumour microenvironment.

Squamous cell carcinoma (SCC) is a treatment-refractory tumour which arises from the epithelium of diverse anatomical sites such as oesophagus, head and neck, lung and skin. Accumulating evidence has revealed a number of genomic, clinical and molecular features commonly observed in SCC of distinct origins. Some of these genetic events culminate in fostering the activity of ΔNp63, a potent oncogene which exerts its pro-tumourigenic effects by regulating specific transcriptional programmes to sustain malignant cell proliferation and survival. In this review, we will describe the genetic and epigenetic determinants underlying ΔNp63 oncogenic activities in SCC, and discuss some relevant transcriptional effectors of ΔNp63, emphasizing their impact in modulating the crosstalk between tumour cells and tumour microenvironment (TME).

ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma.

Accumulating evidence has proved that deregulation of ΔNp63 expression plays an oncogenic role in head and neck squamous cell carcinomas (HNSCCs). Besides p63, the type 1-insulin-like growth factor (IGF) signalling pathway has been implicated in HNSCC development and progression. Most insulin/IGF1 signalling converges intracellularly onto the protein adaptor insulin receptor substrate-1 (IRS-1) that transmits signals from the receptor to downstream effectors, including the PI3K/AKT and the MAPK kinase pathways, which, ultimately, promote proliferation, invasion, and cell survival. Here we report that p63 directly controls IRS1 transcription and cellular abundance and fosters the PI3K/AKT and MAPK downstream signalling pathways. Inactivation of ΔNp63 expression indeed reduces tumour cell responsiveness to IGF1 stimulation, and inhibits the growth potential of HNSCC cells. In addition, a positive correlation was observed between p63 and IRS1 expression in human HNSCC tissue arrays and in publicly available gene expression data. Our findings indicate that aberrant expression of ΔNp63 in HNSSC may act as an oncogenic stimulus by altering the IGF signalling pathway.

HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα.

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.

ΔNp63 regulates the expression of hyaluronic acid-related genes in breast cancer cells.

Triple negative breast cancers (TNBC) represent the most aggressive and clinically relevant breast carcinomas. On the basis of specific molecular signature, the majority of TNBC can be classified as basal-like breast carcinoma. Here, we report data showing that in basal-like breast carcinoma cells ΔNp63 is capable of sustaining the production of the hyaluronic acid (HA), one of the major component of the extracellular matrix (ECM). At molecular level, we found that ΔNp63 regulates the expression of HA-related genes, such as the HA synthase HAS3, the hyaluronidase HYAL-1 and CD44, the major HA cell membrane receptor. By controlling this pathway, ∆Np63 contributes to maintain the self-renewal of breast cancer stem cells. Importantly, high HAS3 expression is a negative prognostic factor of TNBC patients. Our data suggest that in basal-type breast carcinoma ∆Np63 might favor a HA-rich microenviroment, which can sustain tumor proliferation and stemness.

ΔNp63-mediated regulation of hyaluronic acid metabolism and signaling supports HNSCC tumorigenesis.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and several molecular pathways that underlie the molecular tumorigenesis of HNSCC have been identified. Among them, amplification or overexpression of ΔNp63 isoforms is observed in the majority of HNSCCs. Here, we unveiled a ΔNp63-dependent transcriptional program able to regulate the metabolism and the signaling of hyaluronic acid (HA), the major component of the extracellular matrix (ECM). We found that ∆Np63 is capable of sustaining the production of HA levels in cell culture and in vivo by regulating the expression of the HA synthase HAS3 and two hyaluronidase genes, HYAL-1 and HYAL-3. In addition, ∆Np63 directly regulates the expression of CD44, the major HA cell membrane receptor. By controlling this transcriptional program, ∆Np63 sustains the epithelial growth factor receptor (EGF-R) activation and the expression of ABCC1 multidrug transporter gene, thus contributing to tumor cell proliferation and chemoresistance. Importantly, p63 expression is positively correlated with CD44, HAS3, and ABCC1 expression in squamous cell carcinoma datasets and p63-HA pathway is a negative prognostic factor of HNSCC patient survival. Altogether, our data shed light on a ∆Np63-dependent pathway functionally important to the regulation of HNSCC progression.

Setdb1, a novel interactor of ΔNp63, is involved in breast tumorigenesis.

ΔNp63 has been recently involved in self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge of the p63-binding partners potentially involved in the regulation of breast tumour progression is limited. Here, we performed the yeast two hybrid approach to identify p63α interactors involved in breast tumorigenesis and we found that SETDB1, a histone lysine methyl transferases, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. SETDB1 is often amplified in primary breast tumours, and its depletion confers to breast cancer cells growth disadvantage. We identified a list of thirty genes repressed by ΔNp63 in a SETDB1-dependent manner, whose expression is positively correlated to survival of breast cancer patients. These results suggest that p63 and SETDB1 expression, together with the repressed genes, may have diagnostic and prognostic potential.

Anti-tumoral effect of desmethylclomipramine in lung cancer stem cells.

Lung cancer is the most feared of all cancers because of its heterogeneity and resistance to available treatments. Cancer stem cells (CSCs) are the cell population responsible for lung cancer chemoresistance and are a very good model for testing new targeted therapies. Clomipramine is an FDA-approved antidepressant drug, able to inhibit in vitro the E3 ubiquitin ligase Itch and potentiate the pro-apoptotic effects of DNA damaging induced agents in several cancer cell lines. Here, we investigated the potential therapeutic effect of desmethylclomipramine (DCMI), the active metabolite of Clomipramine, on the CSCs homeostasis. We show that DCMI inhibits lung CSCs growth, decreases their stemness potential and increases the cytotoxic effect of conventional chemotherapeutic drugs. Being DCMI an inhibitor of the E3 ubiquitin ligase Itch, we also verified the effect of Itch deregulation on CSCs survival. We found that the siRNA-mediated depletion of Itch induces similar anti-proliferative effects on lung CSCs, suggesting that DCMI might exert its effect, at least in part, by inhibiting Itch. Notably, Itch expression is a negative prognostic factor in two primary lung tumors datasets, supporting the potential clinical relevance of Itch inhibition to circumvent drug resistance in the treatment of lung cancer.